Encapsidated recombinant viral nucleic acid and methods of making and using same

ABSTRACT

The present invention pertains to a method of encapsidating a recombinant poliovirus nucleic acid to obtain a yield of encapsidated viruses which substantially comprises encapsidated recombinant poliovirus nucleic acid. The method of encapsidating a recombinant poliovirus nucleic acid includes contacting a host cell with a recombinant poliovirus nucleic acid which lacks the nucleotide sequence encoding at least a portion of a protein necessary for encapsidation and an expression vector comprising a nucleic acid which encodes at least a portion of one protein necessary for encapsidation under conditions appropriate for introduction of the recombinant poliovirus nucleic acid and the expression vector into the host cell and obtaining a yield of encapsidated viruses which substantially comprises an encapsidated recombinant poliovirus nucleic acid. A foreign nucleotide sequence is generally substituted for the nucleotide sequence of the poliovirus nucleic acid encoding at least a portion of a protein necessary for encapsidation. The invention further pertains to encapsidated recombinant poliovirus nucleic acids produced by the method of this invention and compositions containing the encapsidated or nonencapsidated recombinant poliovirus nucleic acid containing a foreign nucleotide sequence for use in a method of stimulating an immune response in a subject to the protein encoded by the foreign nucleotide sequence.

GOVERNMENT SUPPORT

The work described herein was supported by Public Health Servicecontract (Mucosal Immunology Group) AI 15128, Public Health Servicegrant AI25005 from the National Institutes of Health, and NationalCooperative Vaccine Development Grant (NCVDG) 2 UOI AI28147-06.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/389,459, filed Feb.15, 1995, now U.S. Pat. No. 5,817,512, which is a-continuation-in-partapplication of U.S. Ser. No. 08/087,009, filed Jul. 1, 1993, nowabandoned, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to methods of encapsidating a recombinantviral nucleic acid having a foreign nucleotide sequence substituted forthe nucleotide sequence of the virus encoding at least a portion of aprotein necessary for encapsidation. More particularly, the inventionrelates to methods and compositions for generating an immune response ina subject by using such a recombinant virus.

Live or attenuated viruses have long been used to stimulate the immunesystem in a subject. Poliovirus is an attractive candidate system fordelivery of antigens to the mucosal immune system because of severalbiological features inherent to the virus. First, the pathogenesis ofthe poliovirus is well-studied and the important features identified.The poliovirus is naturally transmitted by an oral-fecal route and isstable in the harsh conditions of the intestinal tract. Primaryreplication occurs in the oropharynx and gastro-intestinal tract, withsubsequent spread to the lymph nodes. Horstmann, D. M. et al. (1959)JAMA170:1-8. Second, the attenuated strains of poliovirus are safe forhumans, and are routinely administered to the general population in theform of the Sabin oral vaccine. The incorporation of foreign genes intothe attenuated strains would be an attractive feature that should poseno more of a health risk than that associated with administration of theattenuated vaccines alone. Third, the entire poliovirus has been cloned,the nucleic acid sequence determined, and the viral proteins identified.An infectious cDNA is also available for poliovirus which has allowedfurther genetic manipulation of the virus. Further, previous studiesusing the attenuated vaccine strains of poliovirus have demonstratedthat a long-lasting systemic and mucosal immunity is generated afteradministration of the vaccine. Sanders, D. Y. and Cramblett, H. G.(1974)J. Ped. 84:406-408; Melnick, J. (1978)Bull. World Health Organ.56:21-38; Racaniello, V. R. and Baltimore, D. (1981)Science 214:916-19;Ogra, P. L. (1984)Rev. Infect. Dis. 6:S361-S368.

Recent epidemiological data suggest that worldwide more than seventypercent of infections with human immunodeficiency virus (HIV) areacquired by heterosexual intercourse through mucosal surfaces of thegenital tract and rectum. Most HIV vaccines developed to date have beendesigned to preferentially stimulate the systemic humoral immune systemand have relied on immunization with purified, whole humanimmunodeficiency virus type 1 (HIV-1) and HIV-1 proteins (Haynes, B. F.(May 1993) Science 260:1279-1286.), or infection with a recombinantvirus or microbe which expresses HIV-1 proteins (McGhee, J. R., andMestecky, J. (1992)AIDS Res. Rev. 2:289-312). A general concern withthese studies is that the method of presentation of the HIV-1 antigen tothe immune system will not stimulate systemic and mucosal tissues togenerate effective immunity at mucosal surfaces. Given the fact that thevirus most often encounters a mucosal surface during sexual (vaginal oranal) transmission, a vaccine designed to stimulate both the systemicand mucosal immune systems is essential. McGhee, J. R., and Mestecky, J.(1992) AIDS Res. Rev. 2:289-312; Forrest, B. D. (1992)AIDS Research andHuman Retroviruses 15 8:1523-1525.

In 1991, a group of researchers reported the construction andcharacterization of chimeric HIV-1-poliovirus genomes. Choi, W. S. etal. (June 1991)J. Virol. 65(6):2875-2883. Segments of the HIV-1 proviralDNA containing the gag, pol, and env gene were inserted into thepoliovirus cDNA so that the translational reading frame was conservedbetween the HIV-1 and poliovirus genes. The RNAs derived from the invitro transcription of the genomes, when transfected into cells,replicated and expressed the appropriate HIV-1 protein as a fusion withthe poliovirus P1 protein. Choi, W. S. et al. (June 1991)J Virol.65(6):2875-2883. However, since the chimeric HIV-1-poliovirus genomeswere constructed by replacing poliovirus capsid genes with the HIV-1gag, pol, or env genes, the chimeric HIV-1-genomes were not capable ofencapsidation after introduction into host cells. Choi, W. S. et al.(June 1991)J. Virol. 65(6):2875-2883. Furthermore, attempts toencapsidate the chimeric genome by cotransfection with the poliovirusinfectious RNA yielded no evidence of encapsidation. Choi, W. S. et al.(June 1991)J. Virol. 65(6):2875-2883.

In 1992, another group of researchers reported the encapsidation of apoliovirus replicon which incorporated the reporter gene,chloramphenicol acetyltransferase (CAT), in place of the region codingfor capsid proteins VP4, VP2, and a portion of VP3 in the genome ofpoliovirus type 3. Percy, N. et al. (Aug. 1992)J. Virol.66(8):5040-5046. Encapsidation of the poliovirus replicon wasaccomplished by first transfecting host cells with the poliovirusreplicon and then infecting the host cells with type 3 poliovirus.Percy, N. et al. (Aug. 1992) J. Virol. 66(8):5040, 5044. The formationof the capsid around the poliovirus genome is believed to be the resultof interactions between capsid proteins and the poliovirus genome.Therefore, it is likely that the yield of encapsidated viruses obtainedby Percy et al. consisted of a mixture of encapsidated poliovirusreplicons and encapsidated nucleic acid from the type 3 poliovirus. Theencapsidated type 3 poliovirus most likely represents a greaterproportion of the encapsidated viruses than does the encapsidatedpoliovirus replicons. The Percy et al. method of encapsidating apoliovirus replicon is, therefore, an inefficient system for producingencapsidated recombinant poliovirus nucleic acid.

Accordingly, it would be desirable to provide a method of encapsidatinga recombinant poliovirus genome which results in a stock of encapsidatedviruses substantially composed of the recombinant poliovirus genome.Such a method would enable the efficient production of encapsidatedpoliovirus nucleic acid for use in compositions for stimulating animmune response to foreign proteins encoded by the recombinantpoliovirus genome.

SUMMARY OF THE INVENTION

The present invention pertains to methods of encapsidating a recombinantpoliovirus nucleic acid to obtain a yield of encapsidated viruses whichsubstantially comprises encapsidated recombinant poliovirus nucleicacid. The methods of encapsidating a recombinant poliovirus nucleic acidinclude providing a recombinant poliovirus nucleic acid which lacks thenucleotide sequence encoding at least a portion of a protein necessaryfor encapsidation and an expression vector lacking an infectiouspoliovirus genome, the nucleic acid of which encodes at least a portionof one protein necessary for encapsidation; contacting a host cell withthe recombinant poliovirus nucleic acid and the expression vector underconditions appropriate for introduction of the recombinant poliovirusnucleic acid and the expression vector into the host cell; and obtaininga yield of encapsidated viruses which substantially comprises anencapsidated recombinant poliovirus nucleic acid. The nucleic acid ofthe expression vector does not interact with the capsid proteins orportions of capsid proteins which it encodes, thereby allowingencapsidation of the recombinant poliovirus nucleic acid and avoidingencapsidation of the nucleic acid of the expression vector. Theinvention further pertains to encapsidated recombinant poliovirusnucleic acids produced by the methods of this invention.

In a preferred embodiment, the methods of encapsidating a recombinantpoliovirus nucleic acid include providing a recombinant poliovirusnucleic acid in which the VP2 and VP3 genes of the P1 capsid precursorregion of the poliovirus genome are replaced by a foreign nucleotidesequence encoding, in an expressible form, a protein or fragmentthereof, such as an immunogenic protein or fragment thereof. Examples ofimmunogenic proteins which can be encoded by thc foreign nucleotidesequence include human immunodeficiency virus type 1 proteins andtumor-associated antigens. A host cell, e.g., a mammalian host cell, isthen contacted with this recombinant poliovirus nucleic acid and anexpression vector lacking an infectious poliovirus genome, such as avaccinia virus, which encodes the poliovirus P1 capsid precursorprotein. Because the expression vector nucleic acid, e.g., vacciniaviral nucleic acid nucleic acid, does not compete with the recombinantpoliovirus nucleic acid for the poliovirus capsid proteins, a yield ofencapsidated viruses which substantially comprises encapsidatedpoliovirus nucleic acid is obtained. Further, the resulting encapsidatedrecombinant poliovirus nucleic acid is able to direct expression of theforeign protein or fragment thereof.

In another preferred embodiment, the methods of encapsidating arecombinant poliovirus nucleic acid include providing a recombinantpoliovirus nucleic acid in which the entire P1 capsid precursor regionof the poliovirus genome is replaced by a foreign nucleotide sequenceencoding, in an expressible form, a protein or fragment thereof, such asan immunogenic protein or fragment thereof. A host cell, e.g., amammalian host cell, is then contacted with this recombinant poliovirusnucleic acid and an expression vector lacking an infectious poliovirusgenome, such as a vaccinia virus, which encodes the poliovirus P1 capsidprecursor protein to thereby generate a yield of encapsidated viruseswhich substantially comprises encapsidated recombinant poliovirusnucleic acid. By these methods of encapsidating recombinant poliovirusnucleic acids, the upper size limit of the foreign nucleotide which canbe inserted into the poliovirus nucleic acid is increased, therebyallowing expression of entire proteins, as well as fragments or portionsof proteins. The present invention also pertains to encapsidatedrecombinant poliovirus nucleic acids which lack the entire P1 capsidprecursor region.

The present invention further pertains to compositions for stimulatingan immune response to an immunogenic protein or fragment thereof and amethod for stimulating the immune response by administering thecompositions to a subject. The compositions typically contain anencapsidated recombinant poliovirus nucleic acid, in a physiologicallyacceptable carrier, which encodes an immunogenic protein or fragmentthereof and directs expression of the immunogenic protein, or fragmentthereof. The compositions are administered to a subject in an amounteffective to stimulate an immune response to the immunogenic protein orfragment thereof, e.g., in an amount effective to stimulate theproduction of antibodies against the immunogenic protein or fragmentthereof in the subject.

The invention still further pertains to methods for generating cellsthat produce a foreign protein or fragment thereof. These methodsinclude contacting host cells with an encapsidated recombinantpoliovirus nucleic acid having a foreign nucleotide sequence substitutedfor the nucleotide sequence which encodes at least a portion of aprotein necessary for encapsidating the recombinant poliovirus nucleicacid and an expression vector lacking an infectious poliovirus genomebut which encodes and directs expression of at least a portion of aprotein necessary for encapsidation of the recombinant poliovirusnucleic acid and directs expression of at least a portion of a proteinnecessary for encapsidating the recombinant poliovirus nucleic acid andmaintaining the cultured host cells under conditions appropriate forintroduction of the recombinant poliovirus nucleic acid and theexpression vector into the host cells, thereby generating modified cellswhich produce a foreign protein or fragment thereof. Such modified cellscan be reintroduced into the subject from which they were obtained tostimulate an immune response in the subject to the foreign protein orfragment thereof produced by the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the translation and proteolytic processingof the poliovirus polyprotein.

FIGS. 2A, 2B, and 2C show chimeric HIV-1-poliovirus genomes containingregions of the HIV-1 gag or pol gene substituted for the poliovirus isP1 gene.

FIG. 3 shows an SDS-polyacrylamide gel on which 3D^(pol) and HIV-1-P1fusion protein expression from cells infected with VV-P1 and transfectedwith recombinant poliovirus RNA was analyzed.

FIGS. 4A, 4B, and 4C show SDS-polyacrylamide gels on which poliovirus-and HIV-1-specific protein expression from cells infected withrecombinant poliovirus RNA which were encapsidated and serially passagedwith capsid proteins provided by VV-P1 were analyzed.

FIG. 5 shows a Northern blot analysis of RNA isolated from a stock ofencapsidated recombinant poliovirus nucleic acid.

FIG. 6 shows an SDS-polyacrylamide gel on which the neutralization ofthe poliovirus nucleic acid encapsidated by VV-P1 with anti-poliovirusantibodies was analyzed.

FIGS. 7A, 7B, and 7C show SDS-polyacrylamide gels on which poliovirus-and HIV-1-specific protein expression from cells infected with a stockof poliovirus nucleic acid encapsidated by type 1 Sabin poliovirus wasanalyzed.

FIGS. 8A, 8B, and 8C show total anti-poliovirus IgG levels in serum frommice after intragastric, intrarectal, and intramuscular administrationof an encapsidated recombinant poliovirus nucleic acid encoding andexpressing at least a portion of the gag protein of humanimmunodeficiency virus type 1.

FIGS. 9A, 9B, and 9C show anti-poliovirus IgA levels in saliva from miceafter intragastric, intrarectal, and intramuscular administration of anencapsidated recombinant poliovirus nucleic acid encoding and expressingat least a portion of the gag protein of human immunodeficiency virustype 1.

FIGS. 10A and 10B show anti-poliovirus IgA in vaginal lavages afterintrarectal, and intramuscular administration of an encapsidatedrecombinant poliovirus nucleic acid encoding and expressing at least aportion of the gag protein of human immunodeficiency virus type 1.

FIGS. 11A, 11B, and 11C show anti-poliovirus IgA in feces from miceafter intragastric, intrarectal, and intramuscular administration of anencapsidated recombinant poliovirus nucleic acid encoding and expressingat least a portion of the gag protein of human immunodeficiency virustype 1.

FIGS. 12A, 12B, and 12C show anti-HIV-1-Gag IgG in serum from mice afterintragastric, intrarectal, and intramuscular administration of anencapsidated recombinant poliovirus nucleic acid encoding and expressingat least a portion of the gag protein of human immunodeficiency virustype 1.

FIGS. 13A, 13B, and 13C show anti-HIV-1-Gag IgA in saliva from miceafter intragastric, intrarectal, and intramuscular administration of anencapsidated recombinant poliovirus nucleic acid encoding and expressingat least a portion of the gag protein of human immunodeficiency virustype 1.

FIGS. 14A and 14B show anti-HIV-1-Gag IgA in vaginal lavages from miceafter intragastric, intrarectal, and intramuscular administration of anencapsidated recombinant poliovirus nucleic acid encoding and expressingat least a portion of the gag protein of human immunodeficiency virustype 1.

FIGS. 15A, 15B, and 15C show anti-HIV-1-Gag IgA in feces from mice afterintragastric, intrarectal, and intramuscular administration of anencapsidated recombinant poliovirus nucleic acid encoding and expressingat least a portion of the gag protein of human immunodeficiency virustype 1.

FIG. 16 shows anti-poliovirus IgG from serum of a Pigtail macaque afterintrarectal administration of an encapsidated recombinant poliovirusnucleic acid encoding and expressing at least a portion of the gagprotein of human immunodeficiency virus type 1.

FIGS. 17A, 17B, and 17C show recombinant poliovirus nucleic acids whichcontain the complete gag gene of HIV-1.

FIGS. 18A and 18B show an analysis of protein expression from cellstransfected with RNA derived from recombinant poliovirus nucleic acidcontaining the gag gene of HIV-1.

FIGS. 19A and 19B show quantitation of recombinant poliovirus RNA fromtransfected cells by Northern blot.

FIG. 20 shows an analysis of poliovirus and HIV-1 specific proteinexpression from cells infected with recombinant poliovirus nucleic acidencapsidated in trails using VV-P1.

FIGS. 21A and 21B show an analysis of protein expression from cellsinfected with normalized amounts of encapsidated recombinant poliovirusnucleic acid stocks and material derived from serial passage ofequivalent amounts of encapsidated recombinant poliovirus nucleic acidvirus stocks with VV-P1.

FIG. 22 shows an analysis of protein expression from cells infected withmaterial derived from the serial passage of encapsidated recombinantpoliovirus nucleic acid with wild-type poliovirus.

FIGS. 23A, 23B, and 23C show construction of recombinant poliovirusnucleic acid containing the gene for carcinoembryonic antigen.

FIGS. 24A and 24B show expression, in transfected cells, ofcarcinoembryonic protein encoded by recombinant poliovirus nucleic acidcontaining the gene for carcinoembryonic antigen.

FIGS. 25A, 25B, and 25C show an analysis of poliovirus andcarcinoembryonic expression from cells infected with recombinantpoliovirus nucleic acid containing the gene for carcinoembryonicantigen; the recombinant poliovirus nucleic acid was encapsidated andserially passaged with capsid proteins provided by VV-P1.

FIGS. 26A and 26B show antibody response to encapsidated recombinantpoliovirus nucleic acid expressing carcinoembryonic antigen.

DETAILED DESCRIPTION OF THE INVENTION

The genome of poliovirus has been cloned and the nucleic acid sequencedetermined. The genomic RNA molecule is 7433 nucleotides long,polyadenylated at the 3' end and has a small covalently attached viralprotein (VPg) at the 5' terminus. Kitamura, N. et al.(1981) Nature(London) 291:547-553; Racaniello, V. R. and Baltimore, D. (1981)Proc.Natl. Acad. Sci. USA 78:4887-4891. Expression of the poliovirus genomeoccurs via the translation of a single protein (polyprotein) which issubsequently processed by virus encoded proteases (2A and 3C) to givethe mature structural (capsid) and nonstructural proteins. Kitamura, N.et al.(1981)Nature (London) 291:547-553; Koch, F. and Koch, G. (1985)The Molecular Biology of Poliovirus (Springer-Verlag, Vienna).Poliovirus replication is catalyzed by the virus-encoded RNA-dependentRNA polymerase (3D^(pol)), which copies the genomic RNA to give acomplementary RNA molecule, which then serves as a template for furtherRNA production. Koch, F. and Koch, G. (1985) The Molecular Biology ofthe Poliovirus (Springer-Verlag, Vienna); Kuhn, R. J. and Wimmer, E.(1987) in D. J. Rowlands et al. (ed.) Molecular Biology of PositiveStrand RNA viruses (Academic Press, Ltd., London).

The translation and proteolytic processing of the poliovirus polyproteinis depicted in FIG. 1 which is a figure from Nicklin, M. J. H. et al.(1986)Bio/Technology 4:33-42. With 25 reference to the schematic in FIG.1, the coding region and translation product of poliovirus RNA isdivided into three primary regions (P1, P2, and P3), indicated at thetop of the figure. The RNA is represented by a solid line and relevantnucleotide numbers are indicated by arrows. Protein products areindicated by waved lines. Cleavage sites are mapped onto the polyprotein(top waved line) as filled symbols; open symbols represent thecorresponding sites which are not cleaved. (∇,∇) are QG pairs, (0,0) areYG pairs, and (⋄,⋄) are NS pairs. Cleaved sites are numbered accordingto the occurrence of that amino-acid pair in the translated sequence.Where the amino acid sequence of a terminus of a polypeptide has beendetermined directly, an open circle has been added to the relevantterminus.

The mature poliovirus proteins arise by a proteolytic cascade whichoccurs predominantly at Q-G amino acid pairs. Kitamura, N. et al.(1981)Nature (London) 291:547-553; Semler, B. L. et al. (1981)Proc.Natl. Acad. Sci. USA 78:3763-3468; Semler, B. L. et al. (1981)Virology114:589-594; Palmenberg, A. C. (1990)Ann. Rev. Microbiol. 44:603-623. Apoliovirus-specific protein, 3C^(pro), is the protease responsible forthe majority of the protease cleavages. Hanecak, R. et al. (1982)Proc.Natl. Acad. Sci. USA:79-3973-3977; Hanecak, R. et al. (1984)Cell37:1063-1073; Nicklin, M. J. H. et al. (1986) Bio/Technology 4:33-42;Harris, K. L et al. (1990)Seminars in Virol. 1:323-333. A second viralprotease, 2A^(pro), autocatalytically cleaves from the viral polyproteinto release P1, the capsid precursor. Toyoda, H. et al. (1986)Cell45:761-770. A second, minor cleavage by 2A^(pro) occurs within the3D^(pol) to give 3C' and 3D'. Lee, Y. F. and Wimmer, E. (1988) Virology166:404-414. Another role of the 2A^(pro) is the shut off of host cellprotein synthesis by inducing the cleavage of a cellular proteinrequired for cap-dependent translation. Bernstein, H. D. et al.(1985)Mol. Cell Biol. 5:2913-2923; Krausslich, H. G. et al. (1987)J.Virol. 61:2711-2718; Lloyd, R. E. et al. (1988)J. Virol. 62:4216-4223.

Previous studies have established that the entire poliovirus genome isnot required for RNA replication. Hagino-Yamagishi, K., and Nomoto, A.(1989)J. Virol. 63:5386-5392. Naturally occurring defective interferingparticles (DIs) of poliovirus have the capacity for replication. Cole,C. N. (1975)Prog. Med. Virol. 20:180-207; Kuge, S. et al. (1986)J. Mol.Biol. 192:473-487. The common feature of the poliovirus DI genome is apartial deletion of the capsid (P1) region that still maintains thetranslational reading frame of the single polyprotein through whichexpression of the entire poliovirus genome occurs. In recent years, theavailability of infectious cDNA clones of the poliovirus genome hasfacilitated further study to define the regions required for RNAreplication. Racaniello, V. and Baltimore, D. (1981)Science 214:916-919.Specifically, the deletion of 1,782 nucleotides of P1, corresponding tonucleotides 1174 to 2956, resulted in an RNA which can replicate upontransfection into tissue culture cells. Hagino-Yamagishi, K. and Nomoto,A. (1989)J. Virol. 63:5386-5392.

Early studies identified three poliovirus types based on reactivity toantibodies. Koch, F. and Koch, G. The Molecular Biology of Poliovirus(Springer-Verlag, Vienna 1985). These three serological types,designated as type I, type II, and type III, have been furtherdistinguished as having numerous nucleotide differences in both thenon-coding regions and the protein coding regions. All three strains aresuitable for use in the present invention. In addition, there are alsoavailable attenuated versions of all three strains of poliovirus. Theseinclude the Sabin type I, Sabin type II, and Sabin type III attenuatedstrains of poliovirus that are routinely given to the population in theform of an oral vaccine. These strains can also be used in the presentinvention.

The recombinant poliovirus nucleic acid of the present invention lacksthe nucleotide sequence encoding at least a portion or a proteinnecessary for encapsidation of the recombinant poliovirus nucleic acid.The nucleotide sequence that is absent from the recombinant poliovirusnucleic acid can be any sequence at least a portion of which encodes atleast a portion of a protein necessary for encapsidation, and the lackof which does not interfere with the ability of the poliovirus nucleicacid to replicate or to translate, in the correct reading frame, thesingle polyprotein through which expression of the entire poliovirusgenome occurs. The recombinant poliovirus nucleic acid can bedeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As the poliovirusgenome is comprised of RNA which replicates in the absence of a DNAintermediate, it is typically introduced into a cell in the form of RNA.This avoids integration of the poliovirus genome into that of the hostcell.

Proteins or portions of proteins necessary for encapsidation of arecombinant poliovirus nucleic acid include, for example, proteins orportions of proteins that are part of the capsid structure. Examples ofsuch proteins are the proteins encoded by the VP 1, VP2, VP3, and VP4genes of the poliovirus P1 capsid precursor region, the Vpg protein, andthose proteins that are necessary for proper processing of structuralproteins of the capsid structure, such as the proteases responsible forcleaving the viral polyprotein.

The nucleotide sequence lacking from the recombinant poliovirus nucleicacid can be the result of a deletion of poliovirus nucleotide sequencesor a deletion of poliovirus nucleotide sequences and insertion of aforeign nucleotide sequence in the place of the deleted sequences.Generally, the nucleotide sequence lacking from the recombinantpoliovirus nucleic acid is the P1 region of the poliovirus genome or aportion thereof, which is replaced by a foreign gene. As used herein,the phrase "which lacks the entire P1 capsid precursor region" when usedto refer to a recombinant poliovirus nucleic acid is intended to includerecombinant poliovirus nucleic acids in which the nucleotide sequenceencoding the P1 capsid precursor protein has been deleted or alteredsuch that the proteins which are normally encoded by this nucleotidesequence are not expressed or are expressed in a form which does notfunction normally. The proteins that are normally encoded by the P1capsid precursor region of the poliovirus genome include the proteinsencoded by the VP1, VP2, VP3, and VP4 genes. A recombinant poliovirusnucleic acid which lacks the entire P1 capsid precursor region,therefore, either does not include a nucleotide sequence which encodesthe proteins encoded by the VP1, VP2, VP3, and VP4 genes or includes anucleotide sequence which encodes, in unexpressible form or inexpressible but not functional form, the proteins encoded by the VP1,VP2, VP3, and VP4 genes. In the present invention, it is specificallycontemplated that recombinant poliovirus nucleic acids which lack theentire P1 capsid precursor region can include nucleotide sequences whichencode amino acids which are included in the proteins encoded by theVP1, VP2, VP3, and VP4 genes so long as the nucleotide sequence encodingthese amino acids of the capsid proteins do not encode the capsidproteins in expressible form or if in expressible form, not functionalform. For example, in one embodiment of the invention, the entire P1capsid precursor region of the poliovirus genome, with the exception ofa nucleotide sequence which encodes the first two amino acids (i.e.,Met-Gly) of the poliovirus P1 capsid precursor protein, is deleted andreplaced with a foreign nucleotide sequence. It is also specificallycontemplated that additional nucleotide sequences from the poliovirusgenome, e.g., nucleotide sequences which encode amino acid sequenceswhich provide cleavage sites for poliovirus enzymes, e.g., 2A protease,or nucleotide sequences which encode other proteins required for properprocessing of a protein encoded by the poliovirus nucleic acid, can beincluded in recombinant poliovirus nucleic acids which lack the entireP1 capsid precursor region.

Additional nucleotide sequences which encode amino acids which are usedas spacers within the poliovirus polyprotein to provide an amino acidsequence of the proper length and of the proper sequence for processingof the poliovirus polyprotein can also be included in recombinantpoliovirus nucleic acids which lack the entire P1 capsid precursorregion.

The foreign nucleotide sequence (or gene) which is substituted for apoliovirus nucleotide sequence preferably is one that encodes, in anexpressible form, a foreign protein or fragment thereof. For example,foreign genes that can be inserted into the deleted region of thepoliovirus nucleic acid can be those that encode immunogenic proteins.Such immunogenic proteins include, for example, tumor-associatedantigens, e.g., human tumor-associated antigens, such ascarcinoembryonic antigen (CEA), the ganglioside antigens GM2, GD2, andGD3 from melanoma cells, the antigen Jen CRG from colorectal and lungcancer cells, synthetic peptides of immunoglobulin epitope from B cellmalignancies, antigens which are products of oncogenes such as erb, neu,and sis, or any other tumor-associated antigen, antigens obtained fromvarious pathogens, such as hepatitis B surface antigen, influenza virushemaglutinin and neuraminidase, human immunodeficiency viral proteins,such as gag, pol, and env, respiratory syncycial virus G protein, andthe VP4 and VP1 proteins of rotavirus, bacterial antigens such asfragments of tetanus toxin, diphtheria toxin, and cholera toxin,mycobactcrium tuberculosis protein antigen B, and urease protein fromHeliobactor pylori. In addition, portions of the foreign genes whichencode immunogenic proteins can be inserted into the deleted region ofthe poliovirus nucleic acid. These genes can encode linear polypeptidesconsisting of B and T cell epitopes. As these are the epitopes withwhich B and T cells interact, the polypeptides stimulate an immuneresponse. It is also possible to insert chimeric foreign genes into thedeleted region of the poliovirus nucleic acid which encode fusionproteins or peptides consisting of both B cell and T cell epitopes.Similarly, any foreign nucleotide sequence encoding an antigen from aninfectious agent can be inserted into the deleted region of thepoliovirus nucleic acid.

The foreign gene inserted into the deleted region of the poliovirusnucleic acid can also encode, in an expressible form, immunologicalresponse modifiers such as interleukins (e.g. interleukin-1,interleukin-2, interleukin-6, etc.), tumor necrosis factor (e.g. tumornecrosis factor-α, tumor necrosis factor-β), or additional cytokines(granulocyte-monocyte colony stimulating factor, interferon-γ). As anexpression system for lymphokines or cytokines, the encapsidatedpoliovirus nucleic acid encoding the lymphokine or cytokine provides forlimited expression (by the length of time it takes for the replicationof the genome) and can be locally administered to reduce toxic sideeffects from systemic administration. In addition, genes encodingantisense nucleic acid, such as antisense RNA, or genes encodingribozymes (RNA molecules with endonuclease or polymerase activities) canbe inserted into the deleted region of the poliovirus nucleic acid. Theantisense RNA or ribozymes can be used to modulate gene expression oract as anti-viral agents. Genes encoding herpes simplex thymidinekinase, which can be used for tumor therapy, SV40 T antigen, which canbe used for cell immortalization, and protein products from herpessimplex virus, e.g., ICP-27, or adeno-associated virus, e.g., Rep, whichcan be used to complement defective viral genomes can be inserted intothe deleted region of the poliovirus nucleic acid.

Foreign genes encoding, in an expressible form, cell surface proteins,secretory proteins, or proteins necessary for proper cellular functionwhich supplement a nonexistent, deficient, or nonfunctional cellularsupply of the protein can also be inserted into the deleted region ofthe poliovirus nucleic acid. The nucleic acid of genes encodingsecretory proteins comprises a structural gene encoding the desiredprotein in a form suitable for processing and secretion by the targetcell. For example, the gene can be one that encodes appropriate signalsequences which provide for cellular secretion of the product. Thesignal sequence can be the natural sequence of the protein or exogenoussequences. In some cases, however, the signal sequence can interferewith the production of the desired protein. In such cases, thenucleotide sequence which encodes the signal sequence of the protein canbe removed. See Example 7, below. The structural gene is linked toappropriate genetic regulatory elements required for expression of thegene product by the target cell. These include a promoter and optionallyan enhancer element along with the regulatory elements necessary forexpression of the gene and secretion of the gene encoded product.

In one embodiment, the foreign genes that are substituted for the capsidgenes of the P1 capsid precursor region of the poliovirus genome are thegag (SEQ ID NO: 3; the sequence of the corresponding gag protein isrepresented by SEQ ID NO: 4), pol (SEQ ID NO: 5; the sequence of thecorresponding pol protein is represented by SEQ ID NO: 6), or env (SEQID NO: 7; the sequence of the corresponding env protein is representedby SEQ ID NO: 8) genes, or portions thereof, of the humanimmunodeficiency virus type 1 (HIV-1). See Example 5, below. Portions ofthese genes are typically inserted in the poliovirus between nucleotides1174 and 2956. The entire genes are typically inserted in the poliovirusbetween nucleotides 743 and 3359. The translational reading frame isthus conserved between the HIV-1 genes and the poliovirus genes. Thechimeric HIV-1-poliovirus RNA genomes replicate and express theappropriate HIV-1-P1 fusion proteins upon transfection into tissueculture. Choi, W. S. et al. (June 1991)J. Virol. 65(6):2875-2883. Inanother embodiment, foreign genes encoding tumor-associated antigens orportions thereof, such as carcinoembryonic antigen or portions thereofcan be substituted for the capsid genes of the P1 capsid precursorregion of the poliovirus genome. See Example 7, below.

Deletion or replacement of the P1 capsid region of the poliovirus genomeor a portion thereof results in a poliovirus nucleic acid which isincapable of encapsidating itself. Choi, W. S. et al. (June 1991)J.Virol. 65(6):2875-2883. Typically, capsid proteins or portions thereofmediate viral entry into cells. Therefore, poliovirus nucleic acid whichis not enclosed in a capsid enters cells on which there is a poliovirusreceptor less efficiently than encapsidated poliovirus nucleic acid. Itis preferred, but not required, therefore, that essential capsidproteins from another source be provided for encapsidation and deliveryof the foreign genes to cells. In the method of this invention,essential poliovirus capsid proteins are provided by an expressionvector which is introduced into the host cell along with the recombinantpoliovirus nucleic acid. The expression vectors can be introduced intothe host cell prior to, concurrently with, or subsequent to theintroduction of the recombinant poliovirus nucleic acid. In analternative embodiment, nonencapsidated recombinant poliovirus nucleicacid can be delivered directly to target cells, e.g., by directinjection into, for example, muscle cells (see, for example, Acsadi etal. (1991)Nature 332: 815-818; Wolff et al. (1990)Science247:1465-1468), or by electroporation, transfection mediated by calciumphosphate, transfection mediated by DEAE-dextran, liposome-mediatedtransfection (Nicolau et al. (1987)Meth. Enz. 149:157-176; Wang andHuang (1987)Proc. Natl. Acad. Sci. USA 84:7851-7855; Brigham et al.(1989)Am. J Med. Sci. 298:278; and Gould-Fogerite et al. (1989)Gene84:429-438), or receptor-mediated nucleic acid uptake (see for exampleWu, G. and Wu, C. H. (1988)J. Biol. Chem. 263:14621; Wilson et al.(1992)J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320), or othermethods of delivering naked nucleic acids to target cells, both in vivoand in vitro, known to those of ordinary skill in the art.

In a preferred method of encapsidating the recombinant poliovirusnucleic acid, the expression vector is introduced into the host cellprior to the introduction of the recombinant poliovirus nucleic acid.The introduction of the expression vector into the host cell prior tothe introduction of the recombinant poliovirus nucleic acid allows theinitial expression of the protein or portion of the protein necessaryfor encapsidation by the expression vector.

Previous studies have established that the replication and expression ofthe poliovirus genes in cells results in the shutoff of host cellprotein synthesis which is accomplished by the 2A^(pro) protein ofpoliovirus. Thus, in order for efficient encapsidation, the expressionvector must express the protein necessary for encapsidation. In orderfor this to occur, the expression vector is generally introduced intothe cell prior to the addition of the recombinant poliovirus nucleicacid.

Expression vectors suitable for use in the present invention includeplasmids and viruses, the nucleic acids of which encode at least aportion of a protein necessary for encapsidation of the recombinantpoliovirus nucleic acid and direct expression of the nucleotide sequenceencoding at least a portion of a protein necessary for encapsidation ofthe recombinant poliovirus nucleic acid. In addition, the nucleic acidof the expression vectors of the present invention does notsubstantially associate with poliovirus capsid proteins or portionsthereof. Therefore, expression vectors of the present invention, whenintroduced into a host cell along with the recombinant poliovirusnucleic acid, result in a host cell yield of encapsidated viruses whichis substantially composed of encapsidated recombinant poliovirus nucleicacid. As used herein, the phrases "substantially composed" or"substantially comprises" when used to refer to a yield of encapsidatedrecombinant poliovirus nucleic acids is intended to include a yield ofencapsidated recombinant poliovirus nucleic acid which is greater than ayield of encapsidated recombinant poliovirus nucleic acid which isgenerated through the use of an expression vector which encodespoliovirus capsid proteins but also includes an infectious poliovirusgenome. Infectious poliovirus genomes can compete with the recombinantpoliovirus nucleic acid for poliovirus capsid proteins, therebydecreasing the yield of encapsidated recombinant poliovirus nucleicacid. Generally, the nucleic acid of the expression vector encodes anddirects expression of the nucleotide sequence coding for a capsidprotein which the recombinant poliovirus nucleic acid is not capable ofexpressing. For example, the expression vector can encode the entire P1capsid precursor protein.

Plasmid expression vectors can typically be designed and constructedsuch that they contain a gene encoding, in an expressible form, aprotein or a portion of a protein necessary for encapsidation of therecombinant poliovirus nucleic acid. Generally, construction of suchplasmids can be performed using standard methods, such as thosedescribed in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual,2nd edition (CSHL Press, Cold Spring Harbor, NY 1989). A plasmidexpression vector which expresses a protein or a portion of a proteinnecessary for encapsidation of the poliovirus nucleic acid isconstructed by first positioning the gene to be inserted (e.g. VP1, VP2,VP3, VP4 or the entire P1 region) after a DNA sequence known to act as apromoter when introduced into cells. The gene to be inserted istypically positioned downstream (3') from the promoter sequence. Thepromoter sequence consists of a cellular or viral DNA sequence which hasbeen previously demonstrated to attract the necessary host cellcomponents required for initiation of transcription. Examples of suchpromoter sequences include the long terminal repeat (LTR) regions ofRous Sarcoma Virus, the origin of replication for the SV40 tumor virus(SV4-ori), and the promoter sequence for the CMV (cytomegalovirus)immediate early protein. Plasmids containing these promoter sequencesare available from a number of companies which sell molecular biologyproducts (e.g. Promega (Madison, Wis.), Stratagene Cloning Systems(LaJolla, Calif.), and Clontech (Palo Alto, Calif.).

Construction of these plasmid expression vectors typically requiresexcision of a DNA fragment containing the gene to be inserted andligation of this DNA fragment into an expression plasmid cut withrestriction enzymes that are compatible with those contained on the 5'and 3' ends of the gene to be inserted. Following ligation of the DNA invitro, the plasmid is transformed into E.coli and the resulting bacteriais plated onto an agar plate containing an appropriate selectiveantibiotic. The E. coli colonies are then grown and the plasmid DNAcharacterized for the insertion of the particular gene. To confirm thatthe gene has been ligated into the plasmid, the DNA sequence of theplasmid containing the insert is determined. The plasmid expressionvector can be transfected into tissue culture cells using standardtechniques and the protein encoded by the inserted gene expressed.

The conditions under which plasmid expression vectors are introducedinto a host cell vary depending on certain factors. These factorsinclude, for example, the size of the nucleic acid of the plasmid, thetype of host cell, and the desired efficiency of transfection. There areseveral methods of introducing the recombinant poliovirus nucleic acidinto the host cells which are well-known and commonly employed by thoseof ordinary skill in the art. These transfection methods include, forexample, calcium phosphate-mediated uptake of nucleic acids by a hostcell and DEAE-dextran facilitated uptake of nucleic acid by a host cell.

Alternatively, nucleic acids can be introduced into cells throughelectroporation, (Neumann, E. et al. (1982)EMBO J. 1:841-845), which isthe transport of nucleic acids directly across a cell membrane by meansof an electric current or through the use of cationic liposomes (e.g.lipofection, Gibco/BRL (Gaithersburg, Md.)). The methods that are mostefficient in each case are typically determined empirically uponconsideration of the above factors.

As with plasmid expression vectors, viral expression vectors can bedesigned and constructed such that they contain a foreign gene encodinga foreign protein or fragment thereof and the regulatory elementsnecessary for expressing the foreign protein. Viruses suitable for usein the method of this invention include viruses that contain nucleicacid that does not substantially associate with poliovirus capsidproteins. Examples of such viruses include retroviruses, adenoviruses,herpes virus, and Sindbis virus. Retroviruses, upon introduction into ahost cell, establish a continuous cell line expressing a foreignprotein. Adenoviruses are large DNA viruses which have a host range inhuman cells similar to that of poliovirus. Sindbis virus is an RNA virusthat replicates, like poliovirus, in the cytoplasm of cells and,therefore, offers a convenient system for expressing poliovirus capsidproteins. A preferred viral expression vector is a vaccinia virus.Vaccinia virus is a DNA virus which replicates in the cell cytoplasm andhas a similar host range to that of poliovirus. In addition, vacciniavirus can accommodate large amounts of foreign DNA and can replicateefficiently in the same cell in which poliovirus replicates. A preferrednucleotide sequence that is inserted in the vaccinia is the nucleotidesequence encoding and expressing, upon infection of a host cell, thepoliovirus P1 capsid precursor polyprotein.

The construction of this vaccinia viral vector is described by Ansardi,D. C. et al. (Apr. 1991)J. Virol. 65(4):2088-2092. Briefly, type 1Mahoney poliovirus cDNA sequences were digested with restriction enzymeNde I, releasing sequences corresponding to poliovirus nucleotides3382-6427 from the plasmid and deleting the P2 and much of the P3encoding regions. Two synthetic oligonucleotides, (5'-TAT-TAG-TAG-ATC-TG(SEQ ID NO: 1)) and 5'-T-ACA-GAT-GTA-CTA-A (SEQ ID NO: 2)) were annealedtogether and ligated into the Nde I digested DNA. The inserted syntheticsequence is places two translational termination codons (TAG)immediately downstream from the codon for the synthetic P1 carboxyterminal tyrosine residue. Thus, the engineered poliovirus sequencesencode an authentic P1 protein with a carboxy terminus identical to thatgenerated when 2A^(pro) releases the P1 polyprotein from the nascentpoliovirus polypeptide. An additional modification was also generated bythe positioning of a Sal I restriction enzyme site at nucleotide 629 ofthe poliovirus genome. This was accomplished by restriction enzymedigest (Ball) followed by ligation of synthetic Sal I linkers. The DNAfragment containing the poliovirus P1 gene was subcloned into thevaccinia virus recombination plasmid, pSC11. Chackrabarti, S. et at.(1985)Mol. Cell Biol. 5:3403-3409. Coexpression of beta-galactosidaseprovides for visual screening of recombinant virus plaques.

The entry of viral expression vectors into host cells generally requiresaddition of the virus to the host cell media followed by an incubationperiod during which the virus enters the cell. Incubation conditions,such as the length of incubation and the temperature under which theincubation is carried out, vary depending on the type of host cell andthe type of viral expression vector used. Determination of theseparameters is well known to those having ordinary skill in the art. Inmost cases, the incubation conditions for the infection of cells withviruses typically involves the incubation of the virus in serum-freemedium (minimal volume) with the tissue culture cells at 37° C. for aminimum of thirty minutes. For some viruses, such as retroviruses, acompound to facilitate the interaction of the virus with the host cellis added. Examples of such infection facilitators include polybrine andDEAE.

A host cell useful in the present invention is one into which both arecombinant poliovirus nucleic acid and an expression vector can beintroduced. Common host cells are mammalian host cells, such as, forexample, HeLa cells (ATCC Accession No. CCL 2), HeLa S3 (ATCC AccessionNo. CCL 2.2), the African Green Monkey cells designated BSC-40 cells,which are derived from BSC-1 cells (ATCC Accession No. CCL 26), andHEp-2 cells (ATCC Accession No. CCL 23). Other useful host cells includechicken cells. Because the recombinant poliovirus nucleic acid isencapsidated prior to serial passage, host cells for such serial passageare preferably permissive for poliovirus replication. Cells that arepermissive for poliovirus replication are cells that become infectedwith the recombinant poliovirus nucleic acid, allow viral nucleic acidreplication, expression of viral proteins, and formation of progenyvirus particles. In vitro, poliovirus causes the host cell to lyse.However, in vivo the poliovirus may not act in a lytic fashion.Nonpermissive cells can be adapted to become permissive cells, and suchcells are intended to be included in the category of host cells whichcan be used in this invention. For example, the mouse cell line L929, acell line normally nonpermissive for poliovirus replication, has beenadapted to be permissive for poliovirus replication by transfection withthe gene encoding the poliovirus receptor. Mendelsohn, C. L. et al.(1989)Cell 56:855-865; Mendelsohn, C. L. et al. (1986)Proc. Natl. Acad.Sci. USA 83:7845-7849.

The encapsidated recombinant poliovirus nucleic acid of the inventioncan be used as a vaccine in the form of a composition for stimulating amucosal as well as a systemic immune response to the foreign proteinencoded and expressed by the encapsidated recombinant poliovirus nucleicacid in a subject. Examples of genes encoding proteins that can beinserted into the poliovirus nucleic acid are described above. Themucosal immune response is an important immune response because itoffers a first line of defense against infectious agents, such an humanimmunodeficiency virus, which can enter host cells via mucosal cells. Atleast a portion of a capsid protein of the encapsidated recombinantpoliovirus nucleic acid is supplied by an expression vector which lacksan infectious poliovirus genome. Expression vectors suitable forsupplying a capsid protein or a portion thereof are described above.Upon administration of the encapsidated recombinant poliovirus nucleicacid, the subject generally responds to the immunizations by producingboth anti-poliovirus antibodies and antibodies to the foreign protein orfragment thereof which is expressed by the recombinant poliovirusnucleic acid. The antibodies produced against the foreign protein orfragment thereof provide protection against the disease or detrimentalcondition caused by the source of the protein or fragment thereof, e.g.,virus, bacteria, or tumor cell. The protection against disease ordetrimental conditions offered by these antibodies is greater than theprotection offered by the subject's immune system absent administrationof the recombinant poliovirus nucleic acids of the invention. Therecombinant poliovirus nucleic acid, in either its DNA or RNA form, canalso be used in a composition for stimulating a systemic and a mucosalimmune response in a subject. Administration of the RNA form of therecombinant poliovirus nucleic acid is preferred as it typically doesnot integrate into the host cell genome.

The encapsidated recombinant poliovirus nucleic acid or thenon-encapsidated recombinant poliovirus nucleic acid can be administeredto a subject in a physiologically acceptable carrier and in an amounteffective to stimulate an immune response to at least the foreignprotein or fragment thereof which is encoded (and its expressiondirected) by the recombinant poliovirus nucleic acid. Typically, asubject is immunized through an initial series of injections (oradministration through one of the other routes described below) andsubsequently given boosters to increase the protection afforded by theoriginal series of administrations. The initial series of injections andthe subsequent boosters are administered in such doses and over such aperiod of time as is necessary to stimulate an immune response in asubject.

Physiologically acceptable carriers suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The composition should typically be sterileand fluid to the extent that easy syringability exists. The compositionshould further be stable under the conditions of manufacture and storageand should be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyetheyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like.

Sterile injectable solutions can be prepared by incorporating theencapsidated recombinant poliovirus nucleic acid in the required amountin an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.

When the encapsidated or nonencapsidated recombinant poliovirus nucleicacid is suitably protected, as described above, the protein can beorally administered, for example, with an inert diluent or anassimilable edible carrier. The protein and other ingredients can alsobe enclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the individual's diet. For oraltherapeutic administration, the active compound can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

As used herein "physiologically acceptable carrier" includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for physiologically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated.

Subjects who can be treated by the method of this invention includeliving organisms, e.g., mammals. Typically, subjects who can be treatedby the method of this invention are susceptible to diseases, e.g.,infectious diseases, cancer, or are susceptible to a detrimentalcondition which can be treated by the methods described herein, e.g., adetrimental condition resulting from a nonexistent, deficient, ornonfunctional supply of a protein which is normally produced in thesubject. Infectious agents which initiate a variety of diseases includemicroorganisms such as viruses and bacteria. Examples of subjectsinclude humans, monkeys, dogs, cats, rats, and mice.

The amount of the immunogenic composition which can stimulate an immuneresponse in a subject can be determined on an individual basis and istypically based, at least in part, on consideration of the activity ofthe specific immunogenic composition used. Further, the effectiveamounts of the immunogenic composition can vary according to the age,sex, and weight of the subject being treated. Thus, an effective amountof the immunogenic composition can be determined by one of ordinaryskill in the art employing such factors as described above using no morethan routine experimentation.

The immunogenic composition is administered through a route which allowsthe composition to perform its intended function of stimulating animmune response to the protein encoded by the recombinant poliovirusnucleic acid. Examples of routes of administration which can be used inthis method include parenteral (subcutaneous, intravenous,intramuscular, intra-arterial, intraperitoneal, intrathecal,intracardiac, and intrasternal), enteral administration (i.e.administration via the digestive tract, e.g. oral, intragastric, andintrarectal administration), and mucosal administration. It is importantto note that the vaccine strains of poliovirus are routinely tested forattenuation by intramuscular and intracerebral injection into monkeys.Thus, it would probably pose no associated health risk if therecombinant poliovirus nucleic acid was given parenterally. Depending onthe route of administration, the immunogenic composition can be coatedwith or in a material to protect it from the natural conditions whichcan detrimentally affect its ability to perform its intended function.

Cells that produce the encapsidated poliovirus nucleic acids of thepresent invention can be introduced into a subject, thereby stimulatingan immune response to the foreign protein or fragment thereof encoded bythe recombinant poliovirus nucleic acid. Generally, the cells that areintroduced into the subject are first removed from the subject andcontacted ex vivo with both the recombinant poliovirus nucleic acid andan expression vector as described above to generate modified cells thatproduce the foreign protein or fragment thereof. The modified cells thatproduce the foreign protein or fragment thereof can then be reintroducedinto the subject by, for example, injection or implantation. Examples ofcells that can be modified by this method and injected into a subjectinclude peripheral blood mononuclear cells, such as B cells, T cells,monocytes and macrophages. Other cells, such as cutaneous cells andmucosal cells can be modified and implanted into a subject. Methods ofintroducing the recombinant poliovirus nucleic acid and the expressionvectors of the invention are described above.

The invention is further illustrated by the following non-limitingexamples. The contents of all references and issued patents citedthroughout this application are expressly incorporated herein byreference.

MATERIALS AND METHODS I:

The following materials and methods were used in Examples 1, 2, 3, and4:

All chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.).Restriction enzymes were obtained from New England Bio-labs (Beverly,Mass.). Tissue culture media was purchased from Gibco/BRL Co.(Gaithersburg, Md). ³⁵ S Translabel (methionine-cysteine) andmethionine-cysteine-free Dulbecco modified Eagle medium (DMEM) werepurchased from ICN Biochemicals (Irvine, Calif.). T7 RNA polymerase wasprepared in this laboratory by the method of Grodberg and Dunn.Grodberg, J. and Dunn, J. J. (1988)J. Bacteriol 170:1245-1253.

Tissue Culture Cells and Viruses

HeLa (human cervical carcinoma) and BSC-40 cells (African green monkeykidney cells) were grown in DMEM supplemented with 5% A-γ newborn calfserum and 5% fetal calf serum (complete medium). The stock of thepoliovirus type 1 Mahoney used in this study was derived fromtransfection of an infectious cDNA clone obtained from B. Semler,University of California at Irvine. Semler, B. L. et al. (1984)NucleicAcids Res. 12:5123-5141. The stock of type 1 Sabin poliovirus wasobtained from the American Type Culture Collection (ATCC Accession No.VR-192). Wild-type vaccinia virus (wt VV) strain WR and the recombinantvaccinia virus VV-P1, which express the poliovirus P1 capsid precursorprotein, have been previously described. Ansardi, D. C. et al. (1991)J.Virol. 65:2088-2092. Antisera to HIV-1 reverse transcriptase (RT) andHIV-1 p25/24 Gag (Steimer, K. S. et al. (1986)Virology 150:283-290) wereobtained through the AIDS Research and Reference Reagent Program(Rockville, Md.). Pooled AIDS patient sera was obtained from the Centerfor AIDS Research, University of Alabama at Birmingham.

In Vitro Transcription Reaction

The in vitro transcription reactions were performed by using T7 RNApolymerase as described previously. Choi, W. S. et al (1991)J. Virol.65:2875-2883. Prior to in vitro transcription, DNA templates werelinearized by restriction enzyme digestion, followed by successivephenol-chloroform (1:1) chloroform extractions and ethanolprecipitation. Reaction mixtures (100 μl) contained 1 to 5 μg oflinearized DNA template, 5×transcription buffer (100 mM Tris [pH 7.7],50 mM MgCl₂, 20 mM spermidine, 250 mM NaCl), 10 mM dithiotheritol, 2mMeach GTP, UTP, ATP, and CTP, 40 U of recombinant RNasin (Promega,Madison, Wis.), and approximately 5μg of purified T7 RNA polymerase perreaction mixture.

After 60 min at 37° C., 5% of the in vitro-synthesized RNA was analyzedby agarose gel electrophoresis.

Encapsidation and Serial Passage of Recombinant Poliovirus Nucleic Acidsby VV-P1

HeLa cells were infected with 20 PFU of VV-P1 (a recombinant virus whichexpresses the poliovirus capsid precursor protein P1) or wild type (wt)VV per cell. After 2 hours of infection, the cells were transfected (byusing DEAE-dextran [500,000 Da] as a facilitator) with RNA transcribedin vitro from the chimeric HIV-1 poliovirus genomes as previouslydescribed. Choi, W. S. et al. (1991)J. Virol. 65:2875-2883. The cultureswere harvested at 24 hours posttransfection. The cells were lysed withTriton X-100 at a concentration of 1%, treated with RNase A, andclarified by low-speed centrifugation at 14,000×g for 20 min at 4° C. asdescribed previously. Li, G. et al. (1991)J. Virol. 65:6714-6723. Thesupernatants were adjusted to 0.25% sodium dodecyl sulfate (SDS),overlaid on a 30% sucrose cushion (30% sucrose, 30 mM Tris [pH 8.0], 1%Triton X-100, 0.1 M NaCl), and centrifuged in a Beckman SW55Ti rotor at45,000 rpm for 1.5h. The pelleting procedure described above has beendemonstrated to be effective for the removal of infectious vacciniavirus to below detectable levels. The supernatant was discarded, and thepellet was washed by recentrifugation for an additional 1.5 hours in alow salt buffer (30 mM Tris [pH8.0], 0.1 M NaCl). The pellets were thenresuspended in complete DMEM and designated passage 1 of the recombinantpoliovirus nucleic acids encapsidated by VV-P 1.

For serial passage of the encapsidated recombinant poliovirus nucleicacids, BSC-40 cells were infected with 20 PFU of VV-P1 per cell. At 2hours postinfection, the cells were infected with passage 1 of theencapsidated recombinant poliovirus nucleic acids. The cultures wereharvested at 24 hours postinfection by three successive freeze-thaws,sonicated, and clarified by centrifugation at 14,000×g for 20 min. Thesupernatants were then stored at -70° C. or used immediately foradditional passages following the same procedure.

Metabolic Labeling and Immunoprecipitation of Viral Proteins

To metabolically label viral proteins from infected-transfected orinfected cells, the cultures were starved for methionine-cysteine at 6hours postinfection by incubation in DMEM minus methionine-cysteine for30 minutes. At the end of this time, ³⁵ S Translabel was added for anadditional hour. Cultures were then processed for immunoprecipitation ofviral proteins by lysing the cells with radioimmunoprecipitation assay(RIPA) buffer (150 mM NaCl, 10 mM Tris [pH 7.8], 1% Triton X-100, 1%sodium deoxycholate, 0.2% SDS). Following centrifugation at 14,000×g for10 min to pellet any debris, designated antibodies were added to thesupernatants, which were incubated at 4° C. rocking for 24 hours. Theimmunoprecipitates were collected by addition of 100 μl of proteinA-Sepharose (10% [wt/vol] in RIPA buffer). After 1 hour of rocking atroom temperature, the protein A-Sepharose beads were collected by briefconfiguration and washed three times with RIPA buffer. The boundmaterial was eluted by boiling for 5 minutes in gel sample buffer (50 mMTris [pH 6.8], 5% SDS, 10% glycerol, 0.01% bromophenol blue, 10%β-mercaptoethanol). The proteins were analyzed by SDS polyacrylamide gelelectrophoresis, and radiolabeled proteins were visualized byfluorography.

Nucleic Acid Hybridization

RNA from a stock of recombinant poliovirus nucleic acids encapsidated byVV-P 1 was analyzed by Northern (RNA) blotting. Stocks of encapsidatedrecombinant poliovirus nucleic acids at passage 14 and a high-titerstock of type 1 Mahoney poliovirus were subjected to RNase A treatmentand overlaid on 30% sucrose cushion (30% sucrose, 30mM Tris [pH 8.0], 1%Triton X-100, 0.1 M NaCl). The samples were centrifuged in a BeckmanSW55Ti rotor at 45,000 rpm for 1.5h. Pelleted virions were resuspendedin TSE buffer (10 mM Tris-HCl [pH 8.0], 50 mM EDTA) and adjusted to 1%SDS and 1% β-mercaptoethanol as previously described. Rico-Hesse, R. etal. (1987)Virology 160:311-322. The resuspended virions were disruptedby extraction three times with phenol-chloroform equilibrated to acidicbuffer and one time with chloroform. The extracted RNA was precipitatedwith 0.2 M LiCl₂, and 2.5 volumes 100% ethanol. The RNA was denaturedand separated on a formaldehyde-agarose gel. The RNA was thentransferred from the gel to a nitrocellulose filter by capillary elution(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndedition (Cold Spring Harbor Laboratory Press, NY)) and cross-linked byusing a UV Stratalinker (Stratagene, LaJolla, Calif.). The conditionsused for prehybridization, hybridization, and washing of RNA immobilizedon filters were previously described (Sambrook, J. et al. (1989)Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring HarborLaboratory Press, NY)). Briefly, the blot was prehybridized inhybridization buffer (50% deionized formamide, 6×SSC [1×SSC is 0.15 MNaCl plus 0.015 M sodium citrate], 1% SDS, 0.1% Tween 20, 100μg of yeasttRNA per ml). The blot was then incubated in hybridization buffercontaining 10⁶ cpm of a [³² p] UTP-labeled riboprobe complementary tonucleotides 671 to 1174 of the poliovirus genome (Choi, W. S. et al(1991) J. Virol. 65:2875-2883) per ml. After hybridization, the blot waswashed two times with 0.1×SSC-0.1% SDS at room temperature and one timeat 65° C. The blot was then exposed to X-ray film with an intensifyingscreen.

Neutralization of The Recombinant Poliovirus Nucleic Acids Encapsidatedby VV-P1 Using Anti-poliovirus Antibodies

For antibody neutralization, encapsidated recombinant poliovirus nucleicacids at passage 9 were pelleted by ultracentrifugation and resuspendedin 250 μl of phosphate-buffered saline (pH 7.0)-0.1% bovine serumalbumin. Samples were preincubated with 25μl of either rabbitanti-poliovirus type 1 Mahoney antisera or preimmune sera per sample at37° C. for 2 hours. Neutralization experiments were conducted on thebasis of the results of preliminary experiments analyzing the capacityof anti-poliovirus antisera to prevent infection of cells by 10⁶ totalPFU of poliovirus under the experimental conditions. The preincubatedsamples were then analyzed for protein expression by infection of BSC-40cells which were metabolically labeled at 6 hours postinfection followedby immunoprecipitation of viral proteins.

Encapsidation of The Recombinant Poliovirus Nucleic Acids by Type 1Sabin Poliovirus

BSC-40 cells were coinfected with 10 PFU of type 1 Sabin poliovirus anda stock of encapsidated recombinant poliovirus nucleic acids (passage14) per cell. The infected cells were harvested at 24 hourspostinfection by three successive freeze-thaws, sonicated and clarifiedby centrifugation at 14,000×g for 20 minutes as described previously(Li, G., et al.

J. Virol. 65:6714-6723). Approximately one-half of the supernatant wasused for serial passaging by reinfection of BSC-40 cells. After 24hours, the cultures were harvested as described above, and the procedurewas repeated for an additional 10 serial passages.

EXAMPLE 1 EXPRESSION OF RECOMBINANT POLIOVIRUS NUCLEIC ACID IN WHICH THEVP2 AND VP3 REGIONS OF THE POLIOVIRUS GENOME ARE REPLACED WITH A PORTIONOF THE HIV-1 GAG OR POL GENES IN CELLS INFECTED WITH AN EXPRESSIONVECTOR WHICH EXPRESSES THE POLIOVIRUS CAPSID PRECURSOR PROTEIN P1

The construction and characterization of recombinant poliovirus nucleicacid in which the HIV-1 gag or pol gene was substituted for VP2 and VP3regions of the poliovirus P1 protein in the infectious cDNA ofpoliovirus have previously been described. Choi, W. S. et al (1991)J.Virol. 65:2875-2883 (FIG. 2). FIG. 2 shows chimeric HIV-1-poliovirusgenomes containing regions of the HIV-1 gag or pol gene substituted forthe poliovirus P1 gene. Details of the construction of plasmidspT7-IC-GAG 1 and pT7-IC-POL have been described by Choi et al. and werepresented as pT7IC-NheI-gag and pT71C-NheI-pol, respectively. Toconstruct pT7-IC-GAG 2, a unique SmaI site was created at nucleotide1580 of the infectious cDNA or poliovirus, and the HIV-1 gag sequenceswere subcloned between nucleotides 1580 and 2470. Insertion of the HIV-1genes maintains the translational reading frame with VP4 and VP1. Invitro transcription from these plasmids generates full-length RNAtranscripts (linearized with SalI). Transfection of full-lengthtranscripts into HeLa cells results in expression of the poliovirus 3CDprotein, a fusion protein between the 3CD and the 3D^(pol) proteins witha molecular mass of 72 kDa. The molecular masses of the HIV-1-P1 fusionproteins are indicated. In previous studies, transfection of thesechimeric RNA genomes into type 1 Mahoney poliovirus-infected cells didnot result in encapsidation of these RNA genomes (Choi, W. S. et al(1991)J. Virol. 65:2875-2883). Under the experimental conditions used,it was possible that the recombinant poliovirus nucleic acid did notefficiently compete with wild-type RNA genomes for capsid proteins. Tocircumvent this problem, a recombinant vaccinia virus (VV-P1) whichexpresses the poliovirus capsid precursor protein P1 upon infection wasused, since recent studies have shown that in cells coinfected withVV-P1 and poliovirus, P1 protein expressed from VV-P1 can enter theencapsidation pathways of wild type poliovirus.

Protein expression from the recombinant poliovirus nucleic acidtransfected into cells previously infected with the recombinant vacciniavirus VV-P1 was analyzed. (FIG. 3) FIG. 3 shows an analysis of 3D^(pol)and HIV-1-P1 fusion protein expression from cells infected with VV-P1and transfected with recombinant poliovirus nucleic acid RNAs. Cellswere infected with VV-P1 at a multiplicity of infection of 20. At 2hours postinfection, cells were transfected with RNA derived from invitro transcription of the designated plasmids. Cells were metabolicallylabeled and cells extracts were incubated with anti-3D^(pol) antibodies(lanes 1 to 5), pooled AIDS patient sera (lanes 6 to 8), or anti-RTantibodies (lane 9), and immunoreactive proteins were analyzed onSDS-polyacrylamide gels. Lanes: 1, cells infected with wild-typepoliovirus: 2 and 6, mock-transfected cells: 3 and 7, cells transfectedwith RNA derived from pT7-IC-GAG 1:4 and 8, cells transfected with RNAderived from pT7-IC-GAG 2; 5 and 9, cells transfected with RNA derivedfrom pT7-IC-POL. The positions of molecular mass standards areindicated. A protein of molecular mass 72 kDa, corresponding to the 3CDprotein of poliovirus, was immunoprecipitated by anti-3D^(pol)antibodies from cells transfected with the recombinant poliovirus RNAbut not from mock-transfected cells. Under the same conditions formetabolic labeling, the 3CD protein, which is a fusion protein betweenthe 3C^(pol) and 3D^(pol) proteins of poliovirus, is predominatelydetected upon incubation of lysates from poliovirus infected cells with3D^(pol) antisera to determine whether the appropriate HIV-1-P1 fusionproteins were also expressed, the extracts were incubated with pooledAIDS patient sera (gag) or rabbit anti-RT antibodies (pol). Expressionof the HIV-1-Gag-P1 fusion proteins corresponding to the predictedmolecular masses 80 and 95 kDa were detected from cells transfected withRNA genomes derived by in vitro transcription of pT7-IC-GAG 1 andpT7-IC-GAG 2, respectively. Similarly, an HIV-1 Pol-P1 fusion protein ofthe predicted molecular mass 85 kDa was immunoprecipitated from cellstransfected with RNA derived from the in vitro transcription ofpT7-IC-POL. These results demonstrate that transfection of therecombinant poliovirus RNA into VV-P1 infected cells results in theexpression of appropriate HIV-1-P1 fusion proteins as well as 3D^(pol)related proteins.

EXAMPLE 2 ENCAPSIDATION AND SERIAL PASSAGE OF RECOMBINANT POLIOVIRUSNUCLEIC ACID IN WHICH THE VP2 AND VP3 REGIONS OF THE POLIOVIRUS GENOMEARE REPLACED WITH A PORTION OF THE HIV-1 GAG OR POL GENES IN CELLS WITHAN EXPRESSION VECTOR WHICH EXPRESSES THE POLIOVIRUS CAPSID PRECURSORPROTEIN P1

In order to determine whether transfection of the recombinant poliovirusnucleic acids encoding the HIV-1 gag and pol genes into VV-P1 infectedcells would result in encapsidation of the recombinant poliovirusnucleic acid, the recombinant poliovirus RNA's were transfected intoeither VV-P1 or wt VV-infected cells, and the encapsidation genomes wereisolated as described in Materials and Methods I. The pelleted materialwas then used to reinfect cells. This procedure was followed bymetabolic labeling of viral proteins and incubation with anti-3D^(pol)or HIV-1- antisera (FIGS. 4A and 4B). FIGS. 4A and 4B show an analysisof poliovirus- and HIV-1-specific protein expression from cells infectedwith recombinant poliovirus nucleic acids which were encapsidated andserially passaged with capsid proteins provided by VV-P1. Cells wereinfected with VV-P1 or wt VV at a multiplicity of infection of 20 andtransfected with RNA derived from in vitro transcription the designatedplasmids. The cells were harvested for isolation of encapsidated genomesas described in Materials and Methods I. The pelleted material was usedto reinfect cells, which were metabolically labeled, and cell lysateswere incubated with the designated antibodies. Immunoreactive proteinswere analyzed on SDS-polyacrylamide gels. FIG. 4A: Lanes: 1 and 5, cellsinfected with pelleted material derived from cells infected with wt VVand transfected with RNA derived from pT7-IC-GAG 1; 2 and 6, cellsinfected with pelleted material derived from cells infected with VV-P1and transfected with RNA derived from pT7-IC-GAG 1; 3 and 7, cellsinfected with pelleted material derived from cells infected with wt VVand transfected with RNA derived from pT7-IC-GAG 2; 4 and 8, cellsinfected with pelleted material derived from cells infected with VV-P1and transfected with RNA derived from pT7-IC-GAG2. FIG. 4B: Lanes: 1 and3, cells infected with pelleted material derived from cells infectedwith wt VV and transfected with RNA derived from pT7-IC-POL; 2 and 4,cells infected with pelleted material derived from cells infected withVV-P1 and transfected with RNA derived from PT7-IC-POL.

The poliovirus 3CD protein was immunoprecipitated from cells infectedwith pelleted material derived from transfection of the recombinantpoliovirus RNA into VV-P1 infected cells. The molecular masses of theHIV-1-P1 fusion proteins immunoprecipitated from the infected cells wereconsistent with the predicted molecular masses and those observed fromexpression of the recombinant poliovirus nucleic acid in transfectedcells (FIG. 2). No 3D^(pol) or HIV-1-P1 proteins were detected fromcells infected with material derived from transfection of the chimericgenomes into wt VV-infected cells, demonstrating a requirement for thepoliovirus P1 protein for encapsidation of the recombinant poliovirusnucleic acid.

To determine whether the encapsidated recombinant poliovirus nucleicacid could be serially passaged, passage 1 stock of the encapsidatedrecombinant poliovirus nucleic acid was used to infect cells that hadbeen previously infected with VV-P1. After 24 hours, the encapsidatedrecombinant poliovirus nucleic acids were isolated as described inMaterials and Methods I and subsequently used to reinfect cells whichhad been previously infected with VV-P1; this procedure was repeated foran additional nine passages. By convention the stocks of seriallypassaged recombinant poliovirus RNA are referred to as vIC-GAG 1,vIC-GAG 2, or vIC-POL. Cells were infected with passage 9 material andmetabolically labeled and the lysates were incubated with antisera topoliovirus 3D^(pol) protein or antibodies to HIV-1 proteins (FIG. 4C).In FIG. 4C, stocks of the encapsidated recombinant poliovirus nucleicacids were also used to infect cells which had been previously infectedwith VV-P1for serial passage of the encapsidated genomes as described inMaterials and Methods I. Cells were infected with serially passagedstocks of recombinant poliovirus nucleic acids at passage 9 andmetabolically labeled, and cell extracts were incubated with thedesignated antibodies (ab). Immunoreactive proteins were analyzed onSDS-polyacrylamide gels. Lanes: 1, cells infected with wild-typepoliovirus; 2 and 5, cells infected with vIC-GAG 1; 3 and 6, Cellsinfected with vIC-GAG2; 4 and 7, cells infected with vIC-POL. Thepositions of molecular mass standards are indicated.

The poliovirus 3CD protein was immunoprecipitated from cells infectedwith poliovirus and the encapsidated recombinant poliovirus nucleicacids. The HIV-1-Gag-P1 and HIV-1-Pol-P1 fusion proteins were alsoimmunoprecipitated from cells infected with the serially passagedrecombinant poliovirus nucleic acids. In contrast, no immunoreactiveproteins were detected from cells which were infected with VV-P1 aloneand immunoprecipitated with the same antisera (FIG. 3).

To determine whether the encapsidated recombinant poliovirus nucleicacids had undergone any significant deletion of genome size as a resultof serial passage with VV-P1, RNA isolated from vIC-GAG 1 at passage 14was analyzed by Northern blotting (FIG. 5). FIG. 5 shows a Northern blotanalysis of RNA isolated from a stock of encapsidated recombinantpoliovirus nucleic acids. Virions were isolated by ultracentrifugationfrom a stock of vIC-GAG 1 at passage 14 and from type 1 Mahoneypoliovirus. The isolated virions were disrupted, and the RNA wasprecipitated, separated in a formaldehyde-agarose gel, and transferredto nitrocellulose. Lanes: 1, RNA isolated from vIC-GAG 1 stock; 2, RNAisolated from poliovirions. Note that the exposure time for the samplein lane 1 of the gel was six times longer than that for lane 2.

For these studies, a riboprobe complementary to nucleotides 671 to 1174of poliovirus, present in the HIV-1-poliovirus chimeric genomes, wasused. RNA isolated from vIC-GAG 1 was compared with RNA isolated fromtype 1 Mahoney poliovirions. The migration of the RNA isolated fromvIC-GAG 1 was slightly faster than that of the wild-type poliovirus RNA,consistent with the predicted 7.0-kb size for RNA from pT7-IC-GAG 1versus the 7.5-kb size for wild-type poliovirus RNA. Furthermore, asingle predominant RNA species from vIC-GAG 1 was detected, indicatingthat no significant deletions of the RNA had occurred during the serialpassages.

Antibody Neutralization of Recombinant Poliovirus Nucleic AcidEncapsidated by VV-P1.

To confirm that the recombinant poliovirus nucleic acid RNA passagedwith VV-P1 was encapsidated in poliovirions, the capacity ofpoliovirus-specific antisera to prevent expression of the HIV-1-P1fusion proteins and poliovirus 3CD protein was analyzed. The results ofthis experiment are important to exclude the possibility that therecombinant poliovirus nucleic acids were being passaged by inclusioninto VV-P1 rather than poliovirions. For these studies, passage 9material of vIC-GAG 1 was preincubated with preimmune type 1 poliovirusantisera as described in Materials and Methods I. After incubation, thesamples were used to infect cells, which were then metabolicallylabeled, and cell lysates were analyzed for expression of poliovirus-and HIV-1 specific proteins after incubation with anti-3D^(pol) antiseraand pooled AIDS patient sera, respectively (FIG. 6). FIG. 6 showsneutralization of recombinant poliovirus nucleic acids encapsidated byVV-P1 with anti-poliovirus antibodies. Cells were infected with apassage 9 stock of vIC-GAG 1 that had been preincubated withanti-poliovirus type 1 antisera or preimmune sera as described inMaterials and Methods I. Infected cells were metabolically labeled, celllysates were incubated with anti-3D^(pol) antibodies (lanes 1 to 3) orpooled AIDS patient sera (lanes 4 and 5), and immunoreactive proteinswere analyzed on SDS-polyacrylamide gels. Lanes: 1, cells infected withwild-type poliovirus (no neutralization); 2 and 4, cells infected withvIC-GAG 1 which had been preincubated with preimmune sera: 3 and 5,cells infected with vIC-GAG 1 which had been preincubated withanti-poliovirus type 1 antisera. The positions of molecular massstandards are indicated.

No expression of the poliovirus 3CD or HIV-1-Gag-P1 fusion protein wasdetected from cells infected with vIC-GAG 1 which had been preincubatedwith the anti-poliovirus antibodies. Expression of 3CD protein andHIV-1-Gag-P1 fusion protein was readily detected from cells infectedwith vIC-GAG 1 which had been preincubated with normal rabbit serum(preimmune). These results demonstrate that the recombinant poliovirusnucleic acids were encapsidated by P1 protein provided in trans by VV-P1which could be neutralized by anti-poliovirus antibodies.

Encapsidation of Serially Passaged Recombinant Poliovirus Nucleic Acidsby Poliovirus

To determine whether the recombinant poliovirus nucleic acid genomescould be encapsidated by P1 protein provided in trans from wild-typepoliovirus, cells were coinfected with type 1 Sabin poliovirus andpassage 14 stock of vIC-GAG 1. After 24 hours, the coinfected cells wereharvested as described in Materials and Methods I, and the extractedmaterial was serially passaged 10 additional times at a highmultiplicity of infection. Cells were infected with passage 10 materialof vIC-GAG 1 and type 1 Sabin poliovirus and metabolically labeled, andcell extracts were incubated with antibodies to type 1 Sabin poliovirus(FIG. 7A), pooled sera from AIDS patients (FIG. 7B), and anti-p24antibodies (FIG. 7C) and the immunoreactive proteins were analyzed onSDS polyacrylamide gels. Lanes: 1, cells infected with type 1 Sabinpoliovirus alone; 2, cells infected with material derived from passage10 of vIC-GAG 1 and type 1 Sabin poliovirus. The positions of relevantproteins are indicated.

Poliovirus capsid proteins were detected from cells infected with type 1Sabin poliovirus alone and from cells infected with material derivedfrom passaging vIC-GAG 1 with type 1 Sabin poliovirus. No HIV-1 specificproteins were detected from cells infected with type 1 Sabin poliovirusalone. A slight cross-reactivity of the HIV-1-Gag-P1 fusion protein withanti-poliovirus antisera was detected in extracts of cells infected withmaterial derived from passaging vIC-GAG 1 with type 1 Sabin poliovirus(FIG. 7A). Although the HIV-1-Gag-P1 fusion protein was clearly detectedfrom cells with type 1 Sabin poliovirus after incubation with pooledAIDS patient sera, some cross-reactivity of the poliovirus capsidproteins were also detected (FIG. 7B). To confirm that the HIV-1-Gag-P1fusion protein had been immunoprecipitated from extracts of cellsinfected with material derived from passaging vIC-Gag 1 with type 1Sabin poliovirus, the extracts were incubated with rabbit anti-p24antiserum (FIG. 7C). Again, detection of the HIV-1-Gag-P1 fusion proteinwas evident from cells infected with material derived from passagingvIC-GAG 1 with type 1 Sabin poliovirus but not from cells infected withtype 1 Sabin alone. Furthermore, HIV-1-Gag-P1 fusion protein expressionwas detected after each serial passage (1 to 10) of vIC-GAG 1 with type1 Sabin poliovirus. These results demonstrate that the chimericrecombinant poliovirus nucleic acids can be encapsidated by P1 proteinprovided in trans from type 1 Sabin poliovirus under the appropriateexperimental conditions and are stable upon serial passage.

EXAMPLE 3 PRODUCTION OF ANTI-POLIOVIRUS AND ANTI-GAG ANTIBODIES IN MICEIMMUNIZED WITH ENCAPSIDATED RECOMBINANT POLIOVIRUS NUCLEIC ACIDCONTAINING A PORTION OF THE HIV-1 GAG GENE

The construction and characterization of chimeric HIV-1 poliovirusnucleic acid in which the HIV-1 gag gene was substituted for VP2 and VP3regions of the poliovirus P1 protein in the infectious cDNA ofpoliovirus was performed as described previously. Choi, W. S. et al.(1991)J. Virol. 65:2875-2883. To evaluate both qualitatively andquantitatively the immune responses against HIV-1 gag expressed fromrecombinant poliovirus nucleic acid, BALB/c mice (5 animals in each ofthree groups) were immunized by parenteral (intramuscular), oral(intragastric) or intrarectal routes. The doses were 2.5×10⁵ virus PFUpoliovirus/mouse for systemic immunization (intramuscular) and 2.5×10⁶PFU poliovirus/mouse for oral immunization. It is important to note thatthe titer refers only to the type II Lansing in the virus preparation,since the encapsidated recombinant poliovirus nucleic acid alone doesnot form plaques due to deletion of the P1 capsids. For oralimmunization, the antigen was resuspended in 0.5 ml of RPMI 1640 andadministered by means of an animal feeding tube (Moldoveanu et al.(1993)J. Infect. Dis. 167:84-90). Intrarectal immunization wasaccomplished by application of a small dose of virus in solution (10μl/mouse intrarectally). Serum, saliva, fecal extract and vaginal lavagewere collected before immunization, and two weeks after the initial doseof the virus.

Collection of Biological Fluids

Biological fluids were collected two weeks after the primaryimmunization, and one week after the secondary immunization. The methodsfor obtaining biological fluids are as follows:

Blood was collected from the tail vein with heparinized glass capillarytubes before and at selected times after immunization. The blood wascentrifuged and plasma collected and stored at -70° C.

Stimulated saliva was collected with capillary tubes after injectionwith carbamyl-choline (1-2μg/mouse). Two μg each of soybean trypsininhibitor and phenylmethylsulfonyl fluoride (PMSF) was added to thesample followed by clarification by centrifugation at 800×g for 15minutes. Sodium azide (0.1% final concentration) and FCS (1% finalconcentration) was added after clarification and the sample stored at-70° C. until the assay.

Vaginal lavages were performed in mice by applying approximately 50 μlsterile PBS into the vagina and then aspirating the outcoming fluid.

Intestinal lavages were performed according to the methods previouslydescribed by Elson et al. (Elson, C.O. et al. (1984)J. Immunol. Meth.67:101-108). For those studies, four doses of 0.5 ml lavage solution(isoosmotic for mouse gastrointestinal secretion) was administered at 15minute intervals using an intubation needle. Fifteen minutes after thelast dose of lavage, 0.1 μg of polycarbine was administered byintraperitoneal injection to the anesthetized mouse. Over the next 10 to15 minutes the discharge of intestinal contents was collected into apetri dish containing a 5 ml solution of 0.1 mg/mil trypsin soybeaninhibitor and 5 mM EDTA. The solid material was removed bycentrifugation (650×g for 10 minutes at 4° C.) and the supernatantcollected. Thirty μl of 100 mM PMSF was then added followed by furtherclarification at 27,000×g for 20 minutes at 4° C. An aliquot of 10 μl of0.1% sodium azide and 10% fetal calf serum was added before storage at-70° C.

Fecal Extract was prepared as previously described (Keller, R., andDwyer, J. E. (1968)J. Immunol. 101:192-202).

Enzyme-Linked Immunoabsorbant Assay

An ELISA was used for determining antigen-specific antibodies as well asfor total levels of immunoglobulins. The assay was performed in 96-wellpolystyrene microtiter plates (Dynatech, Alexandria, Va.). For coating,purified poliovirus (1 μg/well) or HIV specific proteins, or solid phaseadsorbed, and affinity-purified polyclonal goat IgG antibodies specificfor mouse IgG, IgA or IgM (Southern Biotechnology Associates,Birmingham, Ala. (SBA)(1 μg/well)) were employed. Dilutions of serum orsecretions were incubated overnight at 4° C. on the coated and blockedELISA plates and the bound immunoglobulins were detected withhorseradish peroxidase-labeled goat IgG against mouse Ig, IgA, IgG, orIgM (SBA). At the end of the incubation time (3 hours at 37° C.), theperoxidase substrate 2,2-azino bis. (3-ethylbenzthiazoline) sulfonicacid (ABTS) (Sigma, St. Louis, Mo.) in citrate buffer pH 4.2 containing0.0075% H₂ O ₂ was added. The color developed was measured in a TitertekMultiscan photometer (Molecular Devices, Palo Alto, Calif.) at 414 nm.To calibrate the total level of mouse IgA, IgG, IgM levels, purifiedmouse myeloma proteins served as standards. For antigen-specific ELISA,the optical densities were converted to ELISA units, using calibrationcurves obtained from optical density values obtained from referencepools of sera or secretions. The calibration curves were constructedusing a computer program on either 4-parameter logistic or weighedlogit-log models. End point titration values were an alternative way ofexpressing the results. The fold increase values were calculated bydividing post-immunization by pre-immunization values expressed in ELISAunits.

Anti-Poliovirus Antibodies

The levels of anti-poliovirus antibodies were determined by ELISA at Day0 (preimmune), Days 12, and 21 post immunization. A secondadministration of encapsidated recombinant poliovirus nucleic acid wasgiven by the same route at day 21, and samples were collected 14 dayspost to second booster and 45 days post second booster. FIGS. 8A, 8B,and 8C show serum anti-poliovirus antibodies (designated total IgG,representing predominantly IgG, with minor contribution of IgM and IgA)for animals immunized via the intragastric, intrarectal, orintramuscular route. The samples from each of the 5 animals within thegroup were pooled, and the ELISA was used to determine the amounts ofanti-poliovirus antibodies at a 1:20 dilution. A very slight increase inthe anti-poliovirus antibodies present in the serum of mice immunizedvia the intragastric route was observed at Day 45 post boosterimmunization when compared to the pre-immune levels at Day 0. A clearincrease in the serum anti-poliovirus antibodies was observed in theanimals immunized via the intragastric or intramuscular route at Day 14and Day 45 post booster immunization. The levels at Day 14 and 45 postbooster immunization were approximately 5-fold over that observed forthe background levels at Day 0.

In FIGS. 9A, 9B, and 9C, IgA anti-poliovirus antibodies present in thesaliva of animals immunized with the encapsidated recombinant poliovirusnucleic acids were analyzed. In this case, there was a clear increase inthe levels of IgA anti-poliovirus antibodies in animals immunized viathe intragastric, intrarectal, or intramuscular route at Day 14 and 45post booster immunization. In FIGS. 10A and 10B, IgA anti-poliovirusantibodies from the vaginal lavage samples taken from mice immunized viathe intrarectal or intramuscular route were analyzed. In this case,there was a clear increase over the preimmune values at Day 45 postbooster immunization with animals immunized via the intrarectal route.In contrast, there was not a significant increase in the levels of IgAanti-poliovirus antibodies in animals immunized via the intramuscularroute. Finally, as shown in FIGS. 11A, 11B, and 11C, IgA anti-poliovirusantibodies were present in extracts from feces obtained from animalsimmunized via the intragastric, intrarectal or intramuscular route. Inall cases, there was an increase of the IgA anti-poliovirus antibodiesat Day 21, Day 14 post booster immunization and Day 45 post boosterimmunization. Levels were approximately 5-fold over the pre-immunelevels taken at Day 0. It is possible that the levels of anti-poliovirusdetected have been underestimated due to the possibility that theanimals are also shedding poliovirus in the feces at this time. The shedpoliovirus as well as anti-poliovirus antibodies form an immune complexwhich would not be detected in the ELISA assay.

Anti-HIV-1-gag Antibodies

Portions of the same samples that were collected to analyzeanti-poliovirus antibodies were analyzed for the presence ofanti-HIV-1-gag-antibodies. FIGS. 12A, 12B, and 12C show the serum levelsof total IgG (representing IgG as the major species and IgM and IgA asthe minor species) anti-HIV-1-gag antibodies in the serum of animalsimmunized via the intragastric, intrarectal, or intramuscular route. Noconsistent increase in the levels of serum antibodies directed againstHIV-1 -gag antibodies in animals immunized via the intragastric orintrarectal route was observed. This is represented by the fact thatthere was no increase in the levels above that observed at Day 0(pre-immune) value. In contrast, there was an increase in theanti-HIV-1-gag antibodies levels in mice immunized via the intramuscularroute. On Day 21 post immunization, there was a clear increase over thebackground value. The levels of anti-HIV-1-gag antibodies in the serumat Days 14 post boost and 45 post boost were clearly above thepre-immune values in the animals immunized via the intramuscular route.

In FIGS. 13A, 13B, and 13C, IgA anti-HIV-1-gag antibodies present in thesaliva of animals immunized via the intragastric, intrarectal orintramuscular route. In this case, there was a clear increase over thepre-immune levels (Day 0) in animals immunized by all three routes ofimmunization. The highest levels of IgA anti-HIV-1-gag antibodies in thesaliva were found at Day 45 post booster immunization. FIGS. 14A and 14Bshow a similar pattern for the samples obtained from vaginal lavage ofanimals immunized via the intrarectal or intramuscular route. In thisinstance, there was a clear increase at Days 14 and 45 post boosterimmunization in the levels of IgA anti-HIV-1-gag antibodies from animalsimmunized via the intrarectal route of immunization. The animalsimmunized via the intramuscular route exhibited an increase of IgAanti-HIV-1-gag antibodies in vaginal lavage samples starting at Day 12through Day 21. The levels increased following the booster immunizationat Day 21 resulting in the highest levels observed at Day 45 postbooster immunization. In FIGS. 15A, 15B, and 15C, IgA anti-HIV-1 -gagantibodies present in fecal extracts obtained from animals immunized viathe three different routes were analyzed. In general, there was anincrease of the pre-immune levels using all three routes of immunizationthat was most evident at Days 14 and 45 post booster immunization. Theresults of these studies clearly establish that administration of theencapsidated recombinant HIV-1-poliovirus nucleic acids via theintragastric, intrarectal, or intramuscular route results in thegeneration of anti-HIV-1-gag antibodies in serum, saliva, vaginallavage, as well as fecal extracts. A greater serum anti-HIV-1-gagantibody response was obtained by immunization of the animals via theintramuscular route rather than the intragastric or intrarectal routes.However, IgA anti-HIV-1-gag antibodies in secretions of animal immunizedvia all three routes were observed.

EXAMPLE 4 PRODUCTION OF ANTI-POLIOVIRUS ANTIBODIES IN PIGTAIL MACAQUEIMMUNIZED WITH ENCAPSIDATED RECOMBINANT POLIOVIRUS NUCLEIC ACIDCONTAINING A PORTION OF THE HIV-1 GAG GENE

A pigtail macaque was immunized with 5×10⁸ PFU of a virus stock of typeI attenuated poliovirus containing the encapsidated recombinant nucleicacid from pT7IC-Gag #2 (FIG. 2 ). For these studies, intrarectalimmunization was performed because of the high concentration of gutassociated lymphoid tissue in the rectum of primates. The virus wasdeposited in a volume of 1 ml using a syringe filter with soft plastictubing and inserted 1 inch into the rectum. The analysis of theanti-poliovirus and anti-gag antibodies was as described in Example 2except that anti-monkey-specific reagents were substituted foranti-murine-specific reagents.

Serum from the macaque prior to immunization (Day 0), 12 days postprimary immunization (12pp), 27 days post primary immunization (27pp)were collected. A second administration of virus consisting of 1 ml of5×10⁸ PFU given intrarectally and 2.5×10⁷ PFU of virus administeredintranasally at 27 days post primary immunization. Fourteen days afterthe second administration of virus (14 days post booster) serum wascollected.

All serum samples were diluted 1:400 in PBS and the levels of IgGanti-poliovirus antibody were determined by ELISA as described above. Asshown in FIG. 16, there was a clear increase in the serum IgGanti-poliovirus antibodies, as measured by OD₄₁₄ in the ELISA, in theimmunized macaque at 14 days post booster immunization. The levels wereapproximately 10-fold higher than the previous levels (Day 0). Thisstudy shows that intrarectal primary followed by intrarectal-intranasalbooster immunization results in clear increase in the IgGanti-poliovirus antibodies.

MATERIALS AND METHODS II:

The following materials and methods were used in Examples 5 and 6:

All chemicals were purchased from Sigma Chemical Company. Tissue culturemedia and supplements were purchased from Gibco/BRL Company. The [³⁵ S]Translabel (methionine/cysteine) and methionine/cysteine-free DMEM werepurchased from ICN Biochemicals. Restriction enzymes were obtained fromNew England Biolabs. The T7 RNA by the method of Grodberg and Dunn((1988)J. Bacteriol. 170:1245-1253). Synthetic DNA primers were preparedat the University of Alabama Comprehensive Cancer Center facility orobtained from Cruachem, Fisher Co. Tri Reagent-LS was obtained fromMolecular Research Center, Inc.

Tissue Culture Cells and Viruses

HeLa T4 and BSC-40 (African green monkey kidney/cell line derived fromBSC 1 cells) cell monolayers were grown in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 5% fetal calf serum and 1×GMS-Gsupplement (complete medium). The stock of the poliovirus type 1 Mahoneywas derived from transfection of an infectious cDNA clone of poliovirusobtained from B Semler, University of California at Irvine (Semler, B.L. et al. (1984)Nucleic Acids Res. 12:5123-5141). The stock ofpoliovirus type 1 Sabin was obtained from American Type CultureCollection. The recombinant vaccinia virus VV-P1, which expresses thepoliovirus P1 capsid precursor protein upon infection, has also beenpreviously described (Ansardi, D. C. et al. (1991)J. Virol.65:2088-2092). Antisera (recombinant) to HIV-1 p25/24 Gag (Steimer, K.S. et al. (1986)Virol. 150:283-290) and a recombinant vaccinia virusvVK1(Karacostas, V. K. et al. (1989)Proc. Natl. Acad. Sci. (USA)86:8964-8967), which expresses the Pr55^(gag) protein upon infection,was obtained through the AIDS Research and Reference Reagent Program.The antisera to 3D^(pol) has been previously described (Jablonski, S. A.et al. (1991)J. Virol. 65:4565-4572).

Construction of Recombinant Poliovirus Nucleic Acid Containing the HIV-1gag Gene

To subclone the HIV-1 recombinant poliovirus genomes, modifications weremade to the poliovirus cDNA plasmid pT7-IC, which contains thepoliovirus cDNA, and has been described previously (Choi, W. S. et al.(1991)J. Virol. 65:2875-2883). A unique Sac I restriction site wasgenerated at the 5' end of the P1 region in the plasmid pT7-IC by aconservative single base change at nucleotide 748 by site-directedmutagenesis to generate the plasmid pT7-IC-Sac I (Sambrook, J. et al.Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring harbor, New York, 1989). The mutation wasconfirmed by sequence analysis of ds DNA (Sambrook, J. et. al. MolecularCloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York, 1989). A unique SnaBI restrictionsite was then generated in the same plasmid by PCR, at nucleotide 3359,using the following synthetic DNA primers:5'-CAC-CCC-TCT-CCT-ACG-TAA-CCA-AGG-ATC-3'(SEQ ID NO: 9), and5'-GTA-CTG-GTC-ACC-ATA-TTG-GTC-AAC-3'(SEQ ID NO: 10). The amplified DNAfragment was precipitated and digested with SnaBI and BstEII. Afterdigestion of the plasmid pT7-IC-Sac I with SnaBI and BstEII, the PCRfragment was ligated into the plasmid. The resultant plasmid wasdesignated pT7-IC-Sac I-SnaBI.

To construct recombinant poliovirus nucleic acid which contains thecomplete HIV-1 Pr55^(gag) gene, nucleotides 345 to 1837 were amplifiedfrom the plasmid pHXB2 (Ratner, L. et al. (1985)Nature 313:277-284) byPCR using the following DNA primers:5'-GGA-GAG-AGA-TGG-GAG-CTC-GAG-CGT-C-3'(SEQ ID NO: 11), and5'-GCCCCC-CTA-TAC-GTA-TTG-TG-3'(SEQ ID NO: 12). The DNA fragment wasligated into the plasmid pT7-IC-Sac I-SnaBI after digestion of thefragment DNA and pT7-IC-Sac I-SnaBI with Sac I and SnaBI DNA sequencingconfirmed that the translational reading frame was maintained betweenthe foreign gene and poliovirus. The final construct was designated aspT7-IC-Pr55^(gag).

A second recombinant poliovirus nucleic acid containing the HIV-1 gaggene was constructed to position nucleotides 1-949 of the poliovirusgenome 5' to the HIV-1 gag gene. The following primers were designed toamplify a DNA fragment from the plasmid pT7-IC from a unique EcoRI site,located upstream of the T7 RNA polymerase promoter, to nucleotide 949:5'-CCA-GTG-AAT-TCC-TAA-TAC-GAC-TCA-CTA-TAG-GTT-AAA-ACA-GC-3'(SEQ ID NO:13) and5'-CTC-TAT-CCT-GAG-CTCCAT-ATG-TGT-CGA-GCA-GTT-TTT-GGT-TTA-GCA-TTG-3'(SEQID NO: 14). The primers were designed to include a 2A protease cleavagesite (tyrosine-glycine amino acid pair (underlined) preceded by sixwild-type amino acids: Thr-Lys-Asp-Leu-Thr-Thr-Tyr-Gly) (SEQ ID NO: 15),corresponding to the authentic 2A cleavage site in the 3D^(pol) gene atnucleotide 6430 in the poliovirus genome, followed by a Sac Irestriction site at the 3' end of the VP4 gene in the amplifiedfragment. The DNA fragment was ligated into pT7-IC-Pr55^(gag) afterdigestion with EcoRI and Sac I. The final construct was designatedpT7-IC-Pr55^(gag) (VP4/2A).

The construction and characterization of the pT7-IC-Gag 1 has beendescribed in previous studies (Choi, W. S. et al. (1991)J. Virol.65:2875-2883; Porter, D. C. et al. (1993)J. Virol. 67:3712-3719).Briefly, pT7-IC-Gag 1 was constructed by substitution of nucleotides 718to 1549 of the HIV-1 gag gene (amplified using PCR) for the P1 codingregion between nucleotides 1174 and 2470 in the infectious cDNA plasmidpT7-IC. This substitution encompasses most of the VP2 and VP3 capsidsequences while maintaining the VP4 and VP1 coding regions.

Encapsidation and Serial Passage of Recombinant Poliovirus Nucleic AcidContaining the HIV-1 Gag Gene

The encapsidation and serial passage of recombinant poliovirus nucleicacid using VV-P1 has been previously described (Morrow, C.D. et al.(1994) "New Approaches for Mucosal Vaccines for AIDS: Encapsidation andSerial Passage of Poliovirus Replicons that Express HIV-1 Proteins UponInfection" AIDS Res. and Human Retroviruses 10(2); Porter, D.C. et al.(1993)J. Virol. 67:3712-3719). Briefly, HeLa T4 cells were infected with5 PFU/cell of VV-P1, which expresses the poliovirus capsid precursorprotein P1 . At 2 hours post-infection, the cells were transfected usingthe DEAE-Dextran method with RNA transcribed from the chimeric genomesin vitro as previously described (Choi, W. S. et al. (1991)J. Virol.65:2875-2883; Pal-Ghosh, R. et al. (1993)J. Virol. 67:4621-4629; Porter,D.C. et al. (1993)J. Virol. 67:3712-3719). The cultures were harvestedat 24 hours post-transfection by detergent lysis, overlaid on a 30%sucrose cushion (30% sucrose, 30 mM Tris pH 8 0, 1% Triton X-100, 0.1 MNaCI), and centrifuged in a Beckman SW55Ti rotor at 55,000 rpms for 1.5hours (Ansardi, D. C. et al. (1993)J. Virol. 67:3684-3690; Porter, D.C.et al. (1993)J. Virol. 67:3712-3719). The supernatant was discarded andthe pellet washed under the same conditions in a low salt buffer (30mMTris pH 8.0, 0.1 M NaCl) for an additional 1.5 hours. The pellets werethen resuspended in complete DMEM and used for serial passageimmediately or stored at -70° C. until used.

For serial passage of the encapsidated recombinant poliovirus nucleicacid and generation of virus stocks, BSC-40 cells were first infectedwith 10-20 PFU/cell of VV-P1. At 2 hours post-infection, the cells wereinfected with passage 1 of the encapsidated recombinant poliovirusnucleic acid. The cultures were harvested at 24 hours post-infection bythree successive freeze/thaws, sonicated, and clarified by low speedcentrifugation at 14,000×g for 20 minutes. The supernatants were thenstored at -70° C. or used immediately for additional passages followingthe same procedure.

Metabolic Labeling and Immunoprecipitation of Viral Proteins fromInfected Cells

To metabolically label proteins from infected cells, the cultures werestarved for methionine/cysteine at the times indicated post-infection byincubation in DMEM minus methionine/cysteine for 30 minutes. At the endof this time, [³⁵ S] Translabel was added for an additional one hour.Cultures were then processed for immunoprecipitation of viral proteinsby lysing the cells with RIPA buffer (150 mM NaCI, 10 mM Tris pH 7.8, 1%Triton X-100, 1% sodium deoxycholate, 0.2% sodium dodecyl sulfate).Following centrifugation at 14,000×g for 10 minutes, the designatedantibodies were added to the supernatants which were then incubated at4° C. for 24 hours. The immunoprecipitates were collected by addition of100μl protein A-Sepharose (10% weight/volume in RIPA buffer). After a 1hour incubation at room temperature, the protein A-Sepharose beads werecollected by brief centrifugation and washed 3 times with RIPA buffer.The bound material was eluted by boiling 5 minutes in gel sample buffer(62.5 mM Tris pH 6.8, 2% SDS, 20% glycerol, 0.05% bromophenol blue, and0.7M 13-mercaptoethanol). The proteins were analyzed bySDS-polyacrylamide gel electrophoresis and radiolabeled proteins werevisualized by fluorography using sodium salicylate as previouslydescribed (Ansardi, D. C. et al. (1993)J. Virol. 67:3684-3690; Porter,D.C. et al. (1993)J. Virol. 67:3712-3719). The immunoprecipitatedproteins were quantitated by phosphorimagery where indicated (MolecularDynamics).

Nucleic Acid Hybridization of RNA

Total cellular RNA was prepared from cells transfected with equivalentamounts of in vitro transcribed RNA as described by the manufacturerusing Tri Reagent-LS (Molecular Research Center, Inc.). The amounts offull length RNA transcripts were estimated by agarose gelelectrophoresis prior to transfection (Choi, W. S. et al. (1991)J.Virol. 65:2875-1883). The RNA was then denatured, separated on aformaldehyde-1.0% agarose gel, and transferred from the gel to anitrocellulose filter by capillary action. Equivalent amounts of RNA, asmeasured by levels of rRNA, were loaded into each lane of the gel. Foranalysis of encapsidated recombinant poliovirus RNA, the RNA wasisolated from virions (Ricco-Hesse, R. M. et al. (1987)Virol.160:311-322) which had been concentrated through a sucrose cushion aspreviously described (Ansardi, D. C. et al. (1993)J. Virol.67.3684-3690; Porter, D. C. et al. (1993)J. Virol. 67:3712-3719). TheRNA was denatured and spotted onto nitrocellulose using a dot blotapparatus according to established protocols (Sambrook, J. et al.Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1989). The RNA wasimmobilized onto the nitrocellulose by baking in a vacuum oven at 80° C.for 1 hour.

The conditions for prehybridization, hybridization and washing of RNAimmobilized onto nitrocellulose were as described previously (Choi, W.S. et al. (1991)J. Virol. 65:2875-2883; Pal-Ghosh, R. et al. (1993)J.Virol. 67:4621-4629; Porter, D.C. et al. (1993)J. Virol. 67:3712-3719).Briefly, the blot was prehybridized in hybridization buffer (50%deionized formamide, 6×SSC, 1% SDS, 0.1% Tween 20, and 100 μg/mL yeasttRNA). The blot was then incubated in hybridization buffer containing1×10⁶ cpm/mL of a [³² p] labeled riboprobe complementary to nucleotides671-1174 of the poliovirus genome (Choi, W. S. et al. (1991)J. Virol.65:2875-2883; Pal-Ghosh, R. et al. (1993)J. Virol. 67:4621-4629; Porter,D. C. et al. (1993)J. Virol. 67:3712-3719). After hybridization, theblot was washed two times with 0.1×SSC/ 0.1 % SDS at room temperatureand at 65° C. The blot was then exposed to X-ray film with anintensifying screen. The levels of RNA from each sample were quantitatedby phosphorimagery (Molecular Dynamics).

Passage of Recombinant Poliovirus Nucleic Acid Containing the HIV-1 GagGene with Type I Attenuated Poliovirus

Virus stocks of encapsidated recombinant poliovirus nucleic acidcontaining HIV-1 gag gene were serially passaged with wild-typepoliovirus as previously described (Morrow, C. D. et al. (1994) "NewApproaches for Mucosal Vaccines for AIDS: Encapsidation and SerialPassage of Poliovirus Replicons that Express HIV-1 Proteins UponInfection" AIDS Res. and Human Retroviruses 10(2); Porter, D. C. et al.(1993)J. Virol. 67:3712-3719). Briefly, BSC-40 cells were co-infectedwith 10 PFU/cell of type 1 Sabin poliovirus and a virus stock ofencapsidated recombinant poliovirus nucleic acid at pass 21. Theinfected cells were harvested at 24 hours post-infection by threesuccessive freeze/thaws, sonicated, and clarified by low speedcentrifugation. Approximately one-half of the supernatant was used forserial passaging by re-infection of BSC-40 cells. After 24 hours, thecultures were harvested as described above and the procedure wasrepeated for an additional 2 serial passages.

EXAMPLE 5 CONSTRUCTION, EXPRESSION, AND REPLICATION OF RECOMBINANTPOLIOVIRUS NUCLEIC ACIDS CONTAINING THE ENTIRE HIV-1 GAG GENE

To further define the requirements of the P1 region for the replicationand encapsidation of poliovirus RNA, the complete gag gene of HIV-1 wassubstituted for the P1 capsid coding sequences. For these studies theplasmid pT7-IC (FIG. 17A), which contains the promoter sequences for T7RNA polymerase positioned 5' to the complete poliovirus cDNA, was used(Choi, W. S. et al. (1991)J. Virol. 65:2875-2883). A unique Sal Irestriction site is located after the poly (A) tract that can be used tolinearize the cDNA before in vitro transcription; the RNA transcriptsfrom this cDNA are infectious upon transfection into tissue culturecells (Choi, W. S. et al. (1991)J. Virol. 65:2875-2883). In order tosubstitute the entire P1 capsid region with the HIV-1 gag gene, a uniqueSac I restriction site was generated at nucleotide 748, immediatelyfollowing the translational start site of poliovirus. A unique SnaBIrestriction site was generated at nucleotide 3359, which is positionedeight amino acids prior to the 2A protease cleavage site(tyrosine-glycine) located at nucleotide 3386; previous studies havesuggested a requirement for the amino acid at the P4 position forautocatalytic processing of the polyprotein by the 2A protease (Harris,K. et al. (1990)Sem. in Virol. 1:323-333). The resultant plasmid,pT7-IC-Sac I-SnaBI was then used for insertion of the HIV-1 gag gene.pT7-IC-Pr55^(gag) (FIG. 17B) was constructed by insertion of thecomplete HIV-1 gag gene from nucleotides 345 to 1837; the Sac I andSnaBI restriction sites were introduced at the 5' and 3' ends of thegene. Substitution of the entire P1 region from the translational startsite of poliovirus to the 2A protease (3386), which autocatalyticallycleaves from the polyprotein upon translation (Toyoda, H. et al.(1986)Cell 45:761-770), results in expression of Pr55^(gag) proteinafter proteolytic processing of the polyprotein.

Naturally occurring defective interfering (DI) genomes of polioviruscontain heterologous deletions of the P1 coding region that encompassthe VP3, VP3 and VP2 capsid sequences. All known poliovirus Dl genomesmaintain an intact VP4 coding region (Kuge, S. et al. (1986)J. Mol.Biol. 192:473-487). A second recombinant poliovirus nucleic acid wasgenerated in which the gag gene was substituted in frame for the VP2,VP3 and VP1 capsid sequences, from nucleotides 949 to 3359 to maintainthe VP4 coding region. For this construct, a DNA fragment was amplifiedby PCR from the plasmid pT7-IC containing sequences encoding VP4followed by the codons for eight amino acids containing atyrosine-glycine amino acid pair. Substitution of the EcoRI to Sac Ifragment into pT7-IC-Pr55^(gag) results in the final plasmid,pT7-IC-Pr55^(gag) (VP4/2A), which contains the VP4 coding sequencesfused in-frame at the 5' end of the complete gag gene (FIG. 17C). Ineach construct, the insertion of HIV-1 gag gene sequences maintains thetranslational reading frame with poliovirus.

Poliovirus and HIV-1-specific protein expression from the recombinantpoliovirus nucleic acids which contain the HIV-1 gag gene was analyzedafter transfection of recombinant poliovirus RNA into cells which hadbeen previously infected with VV-P1 (FIGS. 18A and 18B). Briefly, Cellswere infected with VV-P1 at a multiplicity of infection of 5. At 2 hourspost infection, the cells were transfected with RNA derived from invitro transcription of the designated plasmids. Cells were metabolicallylabeled, and cell extracts were incubated with the antibodies indicatedand immunoreactive proteins were analyzed on SDS-polyacrylamide gels:(FIG. 18A) Lane 1, mock-transfected cells; Lane 2, cells transfectedwith RNA derived from pT7-IC-Pr55^(gag) ; Lane 3, cells transfected withRNA derived from pT7-IC-Pr55^(gag) (VP4/2A); Lane 4, cells transfectedwith RNA derived from pT7-IC-Gag 1; Lane 5, cells infected with type 1Mahoney poliovirus at a multiplicity of infection of 30. (FIG. 18B):Lane 1, mock-transfected cells; Lane 2, cells transfected with RNAderived from pT7-IC-Pr55^(gag) ; Lane 3, cells transfected with RNAderived from pT7-IC-Pr55^(gag) (VP4/2A); Lane 4, cells infected withvVK1 at a multiplicity of infection of 10; Lane 5, cells transfectedwith RNA derived from pT7-IC-Gag 1. The molecular mass standards andpositions of relevant proteins are indicated.

Under the conditions for metabolic labeling, the 3CD protein, which is afusion between the 3C^(pro) and 3D^(pol) proteins, is the predominant 3Dcontaining viral protein detected from poliovirus-infected cells(Porter, D. C. et al (1993)Virus. Res. 29:241-254). A protein with anapproximate molecular mass of 72 kDa, corresponding to the 3CD proteinof poliovirus, was detected from cells transfected with RNA frompT7-IC-Pr55^(gag) and pT7-IC-Pr55^(gag) (VP4/2A) (FIG. 18A, lanes 2 and3), but not from mock-transfected cells (FIG. 18A, lane 1). The 3CDprotein was also immunoprecipitated from cells transfected with RNAderived from pT7-IC-Gag 1 (FIG. 18A, lane 4), which was used as apositive control for transfections in these studies (Porter, D. C. etal. (1993)J. Virol. 3712-3719).

For analysis of the expression of HIV-1 Gag protein, the extracts wereincubated with antip25/24 antibodies (FIG. 18B). A lysate from cellsinfected with the recombinant vaccinia virus vVK1, which contains theHIV-1 gene sequences encoding the complete gag and pol genes, was usedas a control for Pr55^(gag) protein expression (Karacostas, V. K. et al.(1989)Proc. Natl. Acad. Sci. (USA) 86:8964-8967). A protein with anapparent molecular mass of 55 kDa that co-migrated with proteinimmunoprecipitated from cells infected with vVK1 (FIG. 18B, lane 4) wasdetected from cells transfected with RNA from pT7-IC-Pr55^(gag) andpT7-IC-Pr55^(gag) (VP4/2A) (FIG. 18B, lanes 2 and 3). In addition, aprotein of higher molecular mass was immunoprecipitated from cellstransfected with RNA from pT7-IC-Pr55^(gag) (VP4/2A) (FIG. 18B, lane 3).This protein probably is a VP4-Pr55^(gag) precursor protein.

The replication of the transfected RNA derived from the recombinantpoliovirus nucleic acid was also analyzed by Northern blot (FIGS. 19Aand 19B). HeLa T4 cells were transfected with RNA transcribed in vitrofrom pT7-IC-Pr55^(gag), pT7-IC-Pr55^(gag) (VP4/2A) pT7-IC-Gag 1. At 9hours postransfection, total cellular RNA was prepared, separated in a1% formaldehyde-agarose gel, blotted onto nitrocellulose and analyzedusing a riboprobe complementary to nucleotides 671-1174 of thepoliovirus genome. (Choi, W. S. et al. (1991) J. Virol. 65:2875-2883;Pal-Ghosh, R. et al. (1993)J. Virol. 67:4621-4629; Porter, D. C. et al.(1993)J. Virol. 67:3712-3719) (FIG. 19A) The order of the samples isindicated. The migration of RNA of the predicted size, which Was derivedfrom in vitro transcription of pT7IC-Pr55^(gag) and pT7-IC-Pr55^(gag)(VP4/2A), is indicated by an arrow. The asterisk indicates the migrationof RNA of the expected size which was derived from pT7-IC-Gag 1(Porter,D. C. et al. (1993)J. Virol. 67:3712-3719). Thc radioactivity of theNorthern blot was quantitated using phosphorimagery.

The migration of RNA from pT7-IC-Pr55^(gag) and pT7-IC-Pr55^(gag)(VP4/2A) transfected cells was slightly faster on theformaldehyde-agarose gel than RNA from pT7-IC-Gag 1, which is consistentwith the predicted 6.3-6.4 kb size for RNA from pT7-IC-Pr55^(gag) andpT7-IC-Pr55^(gag) (VP4/2A) versus the 7.0 kb size for RNA frompT7-IC-Gag 1(FIG. 19A). Quantitation of the major bands of radioactivityfrom each sample by phosphorimagery indicated that the values forpT7-IC-Pr55^(gag) and pT7-IC-Pr55^(gag) (VP4/2A) were similar althoughthe amounts of RNA detected from both recombinant poliovirus nucleicacids were lower than that for RNA obtained from pT7-IC-Gag 1 (FIG.19B). Together, these results demonstrate that the RNA frompT7-IC-Pr55^(gag) and pT7-IC-Pr55^(gag) (VP4/2A) replicate to similarlevels in transfected cells.

EXAMPLE 6 ENCAPSIDATION AND SERIAL PASSAGE OF RECOMBINANT POLIOVIRUSNUCLEIC ACID CONTAINING THE ENTIRE HIV-1 GAG GENE

Cells were infected with VV-P1 and then transfected with RNA transcribedin vitro from pT7-IC-Pr55^(gag), pT7-IC-Pr55^(gag) (VP4/2A) andpT7-IC-Gag 1. The encapsidated recombinant poliovirus genomes werepassaged in cells which had been previously infected with VV-P1 for atotal of 21 serial passes. Consistent with the nomenclature used herein,the encapsidated virus stocks of pT7-IC-Pr55^(gag) and pT7-IC-Pr55^(gag)(VP4/2A) are referred to as vIC-Pr55^(gag) and vIC-Pr55^(gag) (VP4/2A),respectively.

For analysis of poliovirus and HIV-1-specific protein expression, pass21 virus stocks of encapsidated recombinant poliovirus nucleic acid wereused to infect cells. After metabolic labeling, lysates from the cellswere incubated with anti-3D^(pol) and anti-p24 antibodies (FIG. 20).With reference to FIG. 20, cells were transfected with RNA derived fromin vitro transcription of the designated plasmids at 2 hourspost-infection with VV-P1. Encapsidated genomes were harvested fromcells as described in Materials and Methods 11 and used to re-infectcells which had been previously infected with VV-P1. The encapsidatedrecombinant poliovirus genomes were subsequently serially passaged inVV-P1-infected cells for 21 serial passes. Cells were infected withvirus stocks at pass 21 and metabolically labeled. Cell lysates wereincubated with the designated antibodies and immunoreactive proteinswere analyzed SDS-polyacrylamide gel; Lanes 1 and 6, mock-infectedcells; Lanes 2 and 7, cells infected with vIC-Pr55^(gag) ; Lanes 3 and8, cells infected with vIC-Pr55^(gag) (VP4/2A); Lanes 4 and 9, cellsinfected with vIC-Gag1; Lane 5, cells infected with type 1 Mahoneypoliovirus; Lane 10, cells infected with vVK1. The molecular massstandards and positions of relevant proteins are indicated.

Although the 3CD protein was detected from each of the recombinantpoliovirus nucleic acid virus stocks, decreased levels of 3CD proteinwere consistently detected from cells infected with virus stocks ofvIC-Pr55^(gag) (FIG. 20, lane 2) as compared to cells infected withvirus stocks of vIC-Pr55^(gag) (VP4/2A) (FIG. 20, lane 3) and vIC-Gag1(FIG. 20, lane 4). Upon incubation of the lysates with anti-p24antibodies, a protein with an apparent molecular mass of 55 kDa wasdetected from the vIC-Pr55^(gag) (FIG. 20, lane 7) and vIC-Pr55^(gag)(VP4/2A) (FIG. 20, lane 8) virus stocks; this protein co-migrated withPr55^(gag) expressed from cells infected with the recombinant vacciniavirus vVK1(FIG. 20, lane 10) (Karacostas, V. et al. (1989)Proc. Natl.Acad. Sci. (USA) 86:8964-8967). Again, infection of cells with thevIC-Pr55^(gag) (VP4/2A) virus stock resulted in an increased level ofthe 55 kDa protein, compared to cells infected with vIC-Pr55^(gag).Consistent with previous studies, vIC-Gag 1 expressed an 80 kDa Gag-P1fusion protein in infected cells (FIG. 20, lane 9) (Porter, D. C. et al.(1993)J. Virol. 67:3712-3719).

Since it has been demonstrated that after transfection that RNA fromeach of the recombinant poliovirus nucleic acids resulted in similarlevels of replication and protein expression, detection of reducedlevels of protein expression from cells infected with vIC-Pr55^(gag) ascompared to vIC-Pr55^(gag) (VP4/2A) could be the result of a differencein infectivity (i.e., interaction with receptor, uncoating) between therecombinant poliovirus nucleic acids. To address this question, RNA wasisolated from equivalent amounts of vIC-Pr55^(gag) and vIC-Pr55^(gag)(VP4/2A) virus stocks, which had been serially passaged with VV-P1 for21 passes. Serial dilutions of the RNA were then spotted ontonitrocellulose and analyzed using a riboprobe as described in Materialsand Methods 11. Quantitation of the radioactivity from each sample byphosphorimagery indicated values from vIC-Pr55^(gag) (VP4/2A) virusstocks which were approximately 15 times higher than the values obtainedfor RNA from vIC-Pr55^(gag). The results of these studies corroboratethe differences in expression of 3CD and HIV-1 Gag protein observed forthe recombinant poliovirus nucleic acids. To address the possibilitythat the recombinant poliovirus nucleic acids might have differences ininfectious potential, cells were infected with equivalent amounts ofencapsidated recombinant poliovirus nucleic acids, as determined by RNAhybridization, and metabolically labeled followed by immunoprecipitationwith anti-3D^(pol) antibodies (FIG. 1A). Equivalent amounts of a 72 kDaprotein, corresponding to the 3CD protein, were detected from each ofthe recombinant poliovirus nucleic acid virus stocks. Quantitation ofthe radioactivity from each sample by phosphorimagery confirmed that thelevels of 3CD were similar.

With reference to FIG. 21A, cells were infected with normalized amountsof encapsidated poliovirus nucleic acid virus stocks and metabolicallylabeled. Cells lysates were incubated with the designated antibodies andimmunoreactive proteins were analyzed on an SDS-polyacrylamide gel: Lane1, mock infected cells; Lane 2, cells infected with vIC-Pr55^(gag)recombinant poliovirus stock; Lane 3, cells infected with vIC-Pr55^(gag)(VP4/2A) recombinant poliovirus stock; Lane 4, cells infected withvIC-Gag1 recombinant poliovirus stock. With reference to FIG. 21B,equivalent amounts of each of the recombinant poliovirus stocks wereserially passaged in VV-P1-infected cells for 2 passes as described inMaterials and Methods II. Cells were infected with material derived frompasses I and 2 and metabolically labeled. Cells lysates were incubatedwith the designated antibodies and immunoreactive proteins were analyzedon an SDS-polyacrylamide gel; Lane U, mock-infected cells; Lane 1, cellsinfected with material from pass 1 of vIC-Pr55^(gag) with VV-P1; Lane 3cells infected with material from pass 1 of vIC-Pr55^(gag) (VP4/2A) withVV-P1; Lane 4, cells infected with material from pass 2 ofvIC-Pr55^(gag) (VP4/2A) with VV-P1; Lane 5, cells infected with materialfrom pass 1 of vIC-Gag 1 with VV-P1; Lane 6, cells infected withmaterial from pass 2 of vIC-Gag 1 with VV-P1; Lane 7, cells infectedwith type 1 Mahoney poliovirus. The molecular mass standards andpositions of relevant proteins are indicated.

To determine whether the decreased levels of RNA isolated from thevIC-Pr55^(gag) virus stock at pass 21 as compared to the vIC-Pr55^(gag)(VP4/2A) and vIC-Gag 1 virus stocks were attributable to differences inthe efficiency of encapsidation of RNA which contains the VP4 codingsequences versus the encapsidation of RNA which has a complete deletionof the P1 region, cells which had been previously infected with VV-P1were infected with normalized amounts of each of the encapsidatedrecombinant poliovirus nucleic acid virus stocks. After 24 hours,complete cell lysis had occurred and the supernatant was processed asdescribed in Materials and Methods II; one additional passage wasperformed in cells previously infected with VV-P1. For analysis ofprotein expression from the serially passaged material, cells wereinfected with material from passages 1 and 2, metabolically labeled, andthe cell lysates were incubated with anti-3D^(pol) antibodies (FIG.21B). Similar amounts of the 3CD protein were detected from each of thepassages of equivalent amounts of vIC-Pr55^(gag) (FIG. 21B, lanes 1 and2), vIC-Pr55^(gag) (VP4/2A) (FIG. 21B, lanes 3 and 4) and vIC-Gag 1recombinant poliovirus nucleic acid virus stocks (FIG. 21B, lanes 5 and6) with VV-P1. Thus, the reduced levels of RNA and 3CD proteinexpression detected from the vIC-Pr55^(gag) recombinant poliovirusnucleic acid virus stocks as compared to vIC-Pr55^(gag) (VP4/2A) andvIC-Gag 1 after 21 serial passes with VV-P1 (FIG. 20) were not apparentafter passage of the recombinant poliovirus nucleic acids with VV-P1 for2 serial passes.

Since all known DIs of poliovirus contain an intact VP4 coding region,it was examined whether the recombinant poliovirus nucleic acid whichcontains the VP4 coding sequences might have an advantage if therecombinant poliovirus nucleic acid had to compete with the wild typegenome for capsid proteins. To determine whether vIC-Pr55^(gag) andvIC-Pr55^(gag) (VP4/2A) could also be maintained upon passage withwild-type poliovirus, cells were co-infected with equal amounts ofeither the vIC-Pr55^(gag), vIC-Pr55^(gag) (VP4/2A) or vIC-Gag 1 and type1 Sabin poliovirus. After 24 hours, complete cell lysis had occurred andthe supernatant was processed as described in Materials and Methods II;two additional passages were performed. Cells were infected withmaterial from each serial passage, metabolically labeled and the cellextracts were incubated with antibodies to p24/25 protein (FIG. 22).With reference to FIG. 22, cells were co-infected with equal amounts ofeither the vIC-Pr55^(gag), vIC-Pr55^(gag) (VP4/2A) or vIC-Gag 1 and type1 Sabin poliovirus. The cells were harvested at 24 hours post-infectionand the supernatant was processed as described in Materials and MethodsII; two additional passages were performed. Cells were infected fromeach of the serial passages and metabolically labeled. The cell lysatesincubated with the designated antibody and immunoreactive proteins wereanalyzed on an SDS-polyacrylamide gel: Lane U, uninfected cells; Lanes1, 2 and 3, cells infected with material derived from the indicatedpasses of vIC-Pr55^(gag) with type 1 Sabin poliovirus; Lanes 4, 5 and 6,cells infected with material derived from the indicated passes ofvIC-PR55^(gag) (VP4/2A) with type 1 Sabin poliovirus; Lanes 7, 8 and 9,cells infected with material derived from the indicated passes ofvIC-Gag 1 with type 1 Sabin poliovirus; Lane PV, cells infected withtype 1 Sabin poliovirus.

Each passage is denoted as follows: P1, pass 1; P2, pass 2; and P3, pass3. The molecular mass standards and positions of relevant proteins areindicated.

No HIV-1-specific protein was cells infected with type 1 Sabinpoliovirus alone (FIG. 22, lane PV); the 80 kDa gag-P1 fusion proteinwas detected from cells infected with material from passages 1, 2 and 3of the vIC-Gag 1 recombinant poliovirus nucleic acid and wild-typepoliovirus (FIG. 22, lanes 7-9) (Porter, D. C. et al. (1993)J. Virol.67:3712-3719).

Upon serial passage of vIC-Pr55^(gag) (FIG. 22, lanes 1-3) andvIC-Pr55^(gag) (VP4/2A) (FIG. 22, lanes 4-6) virus stocks with type 1Sabin, a protein which migrated at approximately 55 kDa was detectedfrom cells infected with material from passages 1, 2, and 3. There wasno consistent difference detected between the levels of Pr55^(gag)expression from either recombinant poliovirus nucleic acid. Thus, thepresence or absence of the VP4 coding region did not effect thecapability of the recombinant poliovirus nucleic acid to compete withthe wild-type poliovirus genomes for the P1 protein that was evidentafter three serial passages.

The construction and characterization of a first poliovirus genome whichcontains the complete 1.5 kb gag gene of HIV-1 substituted for theentire P1 region, and a second poliovirus genome in which the gag geneis positioned 3' to the VP4 coding region of the P1 capsid region aredescribed herein. Transfection of RNA from each of the constructs intocells resulted in similar levels of protein expression and RNAreplication. Both genomes were encapsidated upon transfection into cellspreviously infected with VV-P1. Serial passage of the recombinantpoliovirus nucleic acids with VV-P1 resulted in the production of virusstocks of each of the encapsidated genomes. Analysis of the levels ofencapsidated recombinant poliovirus nucleic acids after extended serialpassage revealed that the recombinant poliovirus nucleic acids whichcontain the VP4 coding region were present at higher levels in theencapsidated virus stocks than the recombinant poliovirus nucleic acidswhich contain the gag gene substituted for the entire P1 region; nodifference was detected in the levels of encapsidation of eitherrecombinant poliovirus genome following limited serial passages in thepresence of VV-P1 or Sabin type 1 poliovirus. The results of this studyare significant because this is the first demonstration that poliovirusgenomes which contain a foreign gene substituted for the entire P1region can be encapsidated by P1 provided in trans.

Although the presence of the VP4 coding region was not absolutelyrequired for RNA encapsidation, it was evident that recombinantpoliovirus nucleic acids which contain a complete substitution of the P1region with the HIV-1 gag gene were encapsidated less efficiently thanrecombinant poliovirus nucleic acids which maintain the VP4 codingsequences (nucleotides 743 to 949) positioned 5' to the gag gene. WhenRNA derived from each of the encapsidated recombinant poliovirus nucleicacid virus stocks after 21 serial passes with VV-P1 was isolated andquantitated by nucleic acid hybridization, the RNA from vIC-Pr55^(gag)(VP4/2A) and vIC-Gag 1 recombinant poliovirus nucleic acid virus stocks,which contained VP4, were present at levels that were 15 and 50 timeshigher, respectively, than RNA from vIC-Pr55^(gag) virus stocks.Although it is clear from these results that VP4 is not required forencapsidation, the presence of VP4 might enhance RNA encapsidation.Since limited passage of equivalent amounts of each of the recombinantpoliovirus nucleic acid virus stocks with VV-P1 indicated no significantdifference in the encapsidation of recombinant poliovirus nucleic acidscontaining VP4 versus recombinant poliovirus nucleic acids which containa deletion of the entire P1 coding region, it was possible that theeffect of VP4 on encapsidation would be more apparent if the recombinantpoliovirus RNA had to compete with the wild-type genomes for the P1capsid protein. This situation would be analogous to the encapsidationof defective interfering (DI) genomes in that the defective genome mustcompete effectively with the wild-type genome to be maintained in thevirus stock. However, it was determined that RNA from vIC-Pr55^(gag) andvIC-Pr55^(gag) (VP4/2A) was maintained in virus stocks for 3 serialpassages in the presence of type 1 poliovirus. Thus, during limitedserial passage the recombinant poliovirus genomes did competeeffectively with type 1 Sabin poliovirus RNA for capsid proteins. Usingthe complementation system described herein, it is possible tosubstitute the entire P1 region with at least 1.5 kb of foreign DNA. Onefeature of the expression system described herein is that the foreignprotein is expressed as a polyprotein which is processed by 2A^(pro).Thus, it is possible to express foreign proteins in a nativeconformation from poliovirus genomes if the residual amino acids at theamino or carboxy termini do not interfere with proper folding.Preliminary experiments have demonstrated the 55 kDa HIV-1 Gag proteinexpressed from poliovirus recombinant poliovirus nucleic acids isbiologically active (i.e. formation of virus-like particles). If theexact protein sequence is required for protein function, the desiredprotein can be expressed using internal ribosomal entry sites positionedwithin the recombinant poliovirus nucleic acid.

MATERIALS AND METHODS III:

The following materials and methods were used in Examples 7, 8, and 9:

Plasmid Constructions

All manipulation of recombinant DNA was carried out according tostandard procedures (Maniatis, T. et al. Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork, 1982). The starting plasmid for these studies, pT7-IC, containsthe entire full-length poliovirus infectious cDNA positioned immediatelydownstream from the phage T7 promoter (Choi, W. S. et al. (1991)J.Virol. 65:2875-2883). The full-length cDNA encoding CEA (shown in SEQ IDNO: 16, the amino acid sequence of CEA is shown in SEQ ID NO: 17),subcloned into pGEM plasmid (Beauchemin, N. et al. (1987)Mol. Cell.Biol. 7:3221-3230), was obtained from Dr. David Curiel, University ofAlabama at Birmingham (originally obtained from Dr. Judy Kantor, NIH,Bethesda, Md.).

For construction of the backbone poliovirus vector used for insertion ofthe carcinoembryonic antigen (CEA) gene, two independent PCR reactionswere performed. The first was used to amplify the region fromnucleotides 1 to 743 of the poliovirus genome using the following PCRprimers: 5'-CCA-GTG-AAT-TCC-TAA-TAC-GAC-TAC-CTA-TAG GTT-AAA-ACA-GC-3'(5'primer) (SEQ ID NO: 18) and 5'-GA-TGA-ACC-CTC-GAGACC-CAT-TAT-G-3'(3'primer) (SEQ ID NO: 19).

A second set of PCR primers were designed to amplify a region of thepoliovirus genome from 3370 to 6117. The PCR primers were designed sothat a unique SnaBI restriction site would be created 12 nucleotidesfrom the end of the P1 gene, resulting in an additional four amino acidsupstream from the tyrosine-glycine cleavage site. For subsequentsubcloning, the PCR product was digested with SnaBI and BglII, whichcuts at nucleotide 5601 in the poliovirus genome. The PCR primers usedwere as follows: 5'-CCA-CCA-AGTACG-TAA-CCA-CAT-ATG-G (5' primer) (SEQ IDNO: 20) and 5'-GTG-AGG-ACTG-CTGG-3'(3' primer) (SEQ ID NO: 21).

The conditions for PCR were as follows: 1 min at 94° C., 3 min at 37°C., and 3 min at 72° C. After 30 cycles, a 7-min incubation at 72° C.was included prior to cessation of the PCR reaction. PCR reactions wereextracted successively with phenol:chloroform (1:1) andchloroform:isoamyl alcohol (24:1), and then DNA was precipitated withethanol. After collection of the precipitate by centrifugation, the DNAwas dried and resuspended in water.

The DNA was then digested with the appropriate restriction endonucleaseenzymes at the 5' and 3' end of the PCR-amplified products.

Construction of pT7-IC-CEA-sig⁻ -

To obtain a signal minus version of the CEA gene, PCR was used toamplify a region from the CEA cDNA. The primers used for this PCRreaction were as follows:5'-CAC-CAC-TGC-CCT-CGA-GAA-GCT-CAC-TAT-TG-3'(5' primer) (SEQ ID NO: 22)and 5'CAC-CAC-TGC-CCT-CGA-GAA-GCT-CAC-TAT-TG-3'(3' primer) (SEQ ID NO:23).

The DNA primers were chosen to create an XhoI site at the 5' end and aSnaBI site at the 3' terminus of the amplified DNA. The length of theamplified DNA was approximately 100 base pairs less than that of thefull-length amplified product for the CEA DNA, corresponding to a lossof 34 amino acids from the amino terminus representing the signalsequence. The conditions for PCR and isolation of the amplified productare as described in Materials and Methods III. Prior to ligation, theamplified product was digested with XhoI and SnaBI.

The plasmid pT7-IC was digested with EcoRI and BglII. The DNA fragmentwhich contains the poliovirus genome from nucleotides 56012 to the SalIsite (1.8 kilobases plus the 3.7 kilobases of the vector=5.5 kilobases)was isolated. In the same ligation, this 5.8-kilobase fragment wasligated with the PCR-amplified products from nucleotides 1-743(EcoRI-XhoI), the CEA gene (XhoI-SnaBI), and the PCR-amplified productcontaining poliovirus nucleotides 3370 (SnaBI) to 5601 (BglII). Afterincubation at 15° C. overnight, the ligated products were transformedinto Escherichia coli DH5α and the colonies were selected onampicillin-containing plates. Plasmids isolated from individual colonieswere screened for the desired insert by restriction enzyme digestion.The final plasmid was designated pT7-IC-CEA-sig⁻.

Cell Culture and Viruses.

HeLa cells were purchased from the American Type Culture Collection andwere maintained in monolayer culture in DMEM (GIBCO/BRL) supplementedwith 5% fetal bovine serum. BSC-40 cells were maintained in DMEM with 5%fetal bovine serum as described previously (Ansardi, D. A. et al.(1991)J. Virol. 65:2088-2092).

The vaccinia viruses used for these studies were grown in TK-143-B cells(American Type Culture Collection) and were concentrated forexperimental use as previously described (Ansardi, D. A. et al. (1991)J.Virol. 65:2088-2092). The titers of vaccinia virus were determined byplaque assay on BSC-40 cell monolayers. The recombinant vaccinia virusused for the encapsidation experiments (VV-P1) was constructed asdescribed previously (Ansardi, D. A. et al. (1991)J. Virol.65:2088-2092). The recombinant vaccinia virus which expresses the CEA(rV-CEA) has been previously described (Kantor, J. et al. (1992)J. Natl.Cancer Inst. 84:1084-1091; Kantor, J. et al. (1992)Cancer Res.52:6917-6925).

In Vitro Transcription, Transfections, and Metabolic Labeling

In vitro transcription was carried out as described previously (Choi, W.S. et al. (1991) J. Virol. 65:2875-2883). The in vitro transcribed RNAwas transfected into HeLa cells with DEAE-dextran (molecular mass, 500kDa) as a facilitator as described previously (Choi, W. S. et al.(1991)J. Virol. 65:2875-2883). The cells were first infected withvaccinia virus for 2 h prior to transfection. After the 2 hour infectionperiod, the cells were washed once with DMEM without methionine-cysteineor leucine (depending on the metabolic label), and incubated in thismedium for an additional 45 min to 1 hour. In the case of recombinantpoliovirus nucleic acid-infected cells, the infections were allowed toproceed 4-6 hours prior to metabolic labeling. For [³⁵S]methionine-cysteine labelings, the cells were washed once andincubated in DMEM without methionine-cysteine plus [³⁵S]methionine-cysteine (Translabel;ICN) 150 μCi/ml final concentration.In the case of metabolic labeling with [³ H]leucine, cells were labeledfor 1.5 h using [³ H]leucine (Amersham) (350 μCi/ml) in a final volumeof 0.2 ml leucine-free DMEM. After the labeling period, the cells werewashed once with PBS and processed for radioimmunoprecipitation asdescribed previously (Ansardi, D. A. et al. (1991)J. Virol65:2088-2092). To detect CEA protein, a CEA-specific monoclonal antibody(Col-1) at a concentration of 3 μg/ml was used.

Encapsidation and Serial Passage of Recombinant poliovirus nucleic acidsby VV-P1

Procedures for encapsidation of the recombinant poliovirus nucleic acidshave been described previously (Porter, D. C. et al. ((1993)J. Virol.67:3712-2719; Ansardi, D. A. et al. (1993)J. Virol. 67:3684-3690).Briefly, HeLa cells were infected with 20 PFUs/cell of VV-P1 for 2hours. The cells were then transfected with in vitro transcribed RNAusing DEAE-dextran (Choi, W. S. et al. (1991)J. Virol. 65:2875-2883).Sixteen hours after transfection, the cells and medium were harvested bydirectly adding Triton X-100 to the medium, at a final concentration of1%. The medium-cell lysate was clarified in a microcentrifuge for 20 minat 14,000×g. The clarified lysate was treated with 20 μg/ml of RNase Aat 37° C. for 15 min, then diluted to 4 ml with 30 mM Tris-HCl (pH 8.0,0.1 M NaCl, 1% Triton X-100), and overlaid on a 0.5 ml-sucrose cushion(30% sucrose, 30 mM Tris-HCl pH 8.0, 1 M NaCl, 0.1% BSA) in SW 55 tubes.The sucrose cushion was centrifuged at 45,000 rpm for 2 h. Pelletedmaterial was washed with PBS-0.1% BSA and recentrifuged at 45,000 rpmfor 2 h. The final pellet was resuspended in 0.6 ml complete medium.BSC-40 cells were infected for 2 hours with 20 PFUs/cell of VV-P1, and0.25 ml of the 0.6 ml was used to infect cells infected with VV-P1;after 24 hours, the cells and media were harvested. This was designatedPass 1.

For serial passage of the encapsidated recombinant poliovirus nucleicacids, BSC-40 cells were infected with 20 PFUs of VV-P1/cell. At 2 hoursposttransfection, the cells were infected with Pass I of theencapsidated recombinant poliovirus nucleic acids. The cultures wereharvested at 24 hours postinfection by three successive freeze-thaws,sonicated, and clarified by centrifugation at 14,000×g for 20 min. Thesupernatants were stored at -70° C. or used immediately for additionalpassages, following the same procedure.

Estimation of the Titer of Encapsidated Recombinant Poliovirus NucleicAcids

Since the encapsidated recombinant poliovirus nucleic acids have thecapacity to infect cells, but lack capsid proteins, they cannot formplaques and therefore virus titers cannot be quantified by traditionalassays. To overcome this problem, a method to estimate the titer of theencapsidated recombinant poliovirus nucleic acids by comparison withwild-type poliovirus of known titer (Porter, D. C. et al. ((1993)J.Virol. 67:3712-2719; Ansardi, D. A. et al. (1993)J. Virol. 67:3684-3690)was used. The resulting titer is then expressed in infectious units ofrecombinant poliovirus nucleic acids, since the infection of cells withthe recombinant poliovirus nucleic acids does not lead to plaqueformation due to the absence of P1 capsid genes. It was determinedexperimentally that the infectivity of equal amounts of infectious unitsof encapsidated recombinant poliovirus nucleic acids correlates withequal amounts of PFUs of wild-type poliovirus.

Immunization of Mice and Analysis of CEA-Specific Antibody Response

The encapsidated recombinant poliovirus nucleic acids contain a type 1Mahoney capsid. Since the type 1 strain of poliovirus does not infectmice, transgenic mice (designated as Tg PVR1) which express the receptorfor poliovirus and are susceptible to poliovirus and are susceptible topoliovirus infection (Ren, R. et al. (1990)Cell 63:353-362) were used.

Mice (4-5-week old) were immunized by i.m. infection at monthlyintervals with recombinant poliovirus nucleic acids expressing CEA; eachmouse received 3 doses containing approximately 3-10⁴ infectiousunits/mouse in 50 μl sterile PBS. To remove residual VV-P1, therecombinant poliovirus nucleic acid preparations were incubated withanti-vaccinia virus antibodies (Lee Biomolecular, San Diego, Calif.).The complete removal of residual VV-P1 was confirmed by the lack ofvaccinia virus plaques after a 3-day plaque assay. Blood was collectedfrom the tail veins of mice before and at selected times afterimmunization, centrifuged, and the plasma was collected and frozen untilassay. ELISA was used for the determination of antigen-specificantibodies. The assays were performed in 96-well polystyrene microtiterplates (Dynatech, Alexandria, Va.) coated with recombinant CEA or wholepoliovirus type 1 at a concentration of 5 and 1 μg/ml, respectively. TheCEA used for these studies was expressed in E. coli, using a pET vectorwith a 6-histidine affinity tag to facilitate purification (Novagen).The majority of the CEA product isolated from the nickel column used forpurification was an 80-kDa protein corresponding to the nonglycosylatedCEA. The poliovirus type 1 (Sabin) used was grown in tissue culturecells and purified by centrifugation (Ansardi, D. A. et al. (1993)J.Virol. 67:3684-3690). Dilutions of sera were incubated overnight at 4°C. on coated and blocked ELISA plates, and the bound immunoglobulinswere detected with horseradish peroxidase-labeled antimouseimmunoglobulins (Southern Biotechnology Associates, Birmingham, Ala.).At the end of the incubation time (3 hours at 37° C.), the peroxidasesubstrate 2,2'-azino-bis-(3ethylbenzthiazoline) sulfonic acid (Sigma,St. Louis, Mo.) in citrate buffer (pH 4.2) containing 0.0075% H₂ O₂ wasadded. The color developed was measured in V_(max) kinetic microplatereader (Molecular Devices, Palo Alto, Calif.) at 414 nm. The resultswere expressed as absorbance values at a fixed dilution or as end pointtitration values.

EXAMPLE 7 CONSTRUCTION OF RECOMBINANT POLIOVIRUS NUCLEIC ACID CONTAININGTHE GENE FOR CARCINOEMBRYONIC ANTIGEN

The starting plasmid for the experiments described herein contains thefull-length infectious poliovirus cDNA positioned downstream from aphage T7 promoter, designated pT7-IC (Choi, W. S. et al. (1991)J. Virol.65:2875-2883) (FIG. 23A). With reference to FIG. 23A, the polioviruscapsid proteins (VP4, VP3, VP2, and VP1) are encoded in the P1 region ofthe poliovirus genome; the viral proteinase 2A and viral proteins 2B and2C are encoded in the P2 region; and the viral proteins 3AB, 3C, and 3D(RNA polymerase) are encoded in the P3 region. The relevant restrictionsites used for construction of the recombinant poliovirus nucleic acidcontaining the gene for CEA are indicated. With reference to FIG. 23B,which is a schematic of the CEA protein, the signal sequence of the CEAprotein consists of 34 amino acids (black box). The signal peptidasecleavage site occurs between the alanine and lysine amino acids. Thecodon for the carboxyl terminal isoleucine amino acid is followed by aTAA termination codon. Construction of the recombinant poliovirusnucleic acid containing the signal-minus CEA gene occurred as follows:PCR was used to amplify the CEA-gene encoding amino acids from thelysine at the amino terminus of signal-minus CEA to the isoleucine atthe COOH terminus of CEA as shown in FIG. 23B. To subclone the geneencoding the signal-minus CEA protein, XhoI and SnaBI restrictionendonuclease sites were positioned within the PCR primers. The finalconstruct encodes the first two amino acids of the poliovirus P1 protein(Met-Gly) followed by two amino acids, leucine and glutamic acid(encoded by the XhoI restriction site) followed by the lysine amino acidof the signal-minus CEA protein. The CEA gene was positioned so thatnine amino acids will be spaced between the C-terminal isoleucine of CEAand the tyrosine-glycine cleavage site for the 2A proteinase; theleucine amino acid required for 2A cleavage is boxed in FIG. 23C. Thisfinal construct, as shown in FIG. 23C, was designated pT7-IC-CEA-sig⁻.

After the pT7-IC plasmid is linearized at the unique Sal I restrictionsite, in vitro transcription mediated by phage T7 RNA polymerase is usedto generate RNA transcripts for transfection. Transfection of the invitro RNA transcript into tissue culture cells (i.e., HeLa cells)results in translation and replication of the RNA, which leads toproduction of infectious poliovirus. It has been found that theinfectivity of the RNA derived from this plasmid is in the range of 10⁶PFUs/μg transfected RNA (Choi, W. S. et al. (1991)J. Virol.65:2875-2883). Previous studies have found that the majority of the P1region of the poliovirus cDNA can be deleted without affecting thecapacity of the resulting RNA genome to replicate when transfected intocells (Kaplan, G. et al. (1988)J. Virol. 62:1687-1696). To extend thesestudies, it was investigated whether the entire P1 region can besubstituted with the 2.4-kilobase cDNA for CEA (FIG. 23B; Beauchemin, N.et al. (1987)Mol. Cell. Biol. 7:3221-3230; Oikawa, S. et al.(1987)Biochim. Biophys. Acta. 142:511-518).

In preliminary studies, it was found that RNA containing full-length CEAwas not replication competent. It was possible that the signal sequence(amino acids 1-34) of the CEA protein was directing the CEA-P2-P3 fusionprotein to the endoplasmic reticulum and in doing so preventedreplication of the RNA. To test this possibility, the CEA gene wasengineered to remove the first 34 amino acids of the CEA protein, whichhas been postulated to be the signal sequence (Oikawa, S. et al.(1987)Biochim. Biophys. Acta. 142:511-518; Thompson, J. et al.(1988)Tumor Biol. 9:63-83). PCR was used to amplify a region from aminoacids 35-688 of the CEA gene that was then subcloned into the poliovirusrecombinant poliovirus nucleic acid. The resulting DNA encoded the firsttwo amino acids of the poliovirus P1 protein (Met-Gly) followed by twoamino acids (Leu-Glu) derived from the XhoI restriction endonucleasesite, followed by amino acid 35 (Lys) of the CEA protein. The isoleucinein CEA was fused to an additional nine amino acids(Tyr-Val-Thr-Lys-Asp-Leu-Thr-Thr-Tyr) in the predicted protein product.In this CEA protein, a leucine residue at the P4 position was includedfor optimal 2A autocatalytic cleavage (Harris, K. S. et al. (1 990)Semin. Virol. 1:323-333).

Following in vitro transcription of pT7-IC-CEA-sig⁻, the RNA transcriptswere transfected into cells previously infected with VV-P1. For thesestudies five independent clones containing the signal-minus CEA gene(designated as sig⁻ CEA) were tested. As a positive control, arecombinant poliovirus nucleic acid which contains the HIV-1 gag gene(corresponding to the capsid, p24 protein) positioned betweennucleotides 1174 and 2470 of the poliovirus genome was used. Cells werealso infected with poliovirus to serve as a control in theseexperiments. At 6 hours posttransfection, the cells were metabolicallylabeled and ³⁵ S-labeled proteins were immunoprecipitated with eitheranti-3D^(pol) (FIG. 24A) of anti-CEA (Col-1 monoclonal antibody (FIG.24B). The immunoprecipitated proteins were separated on SDS-10%polyacrylamide gels, and autoradiograms of these gels were generated(shown in FIGS. 24A and 24B). Additional sets of cells were eitherinfected with poliovirus (FIG. 24A) or a recombinant vaccinia viruswhich expresses CEA (rV-CEA, FIG. 24B) to serve as a source of markerproteins. The origins of the samples in each of the lanes for both FIG.24A and FIG. 24B are as follows: Lane 1, mock transfected cells; Lane 2,cells transfected with RNA derived from clone 1 of PT7-IC-CEA-sig⁻ ;Lane 3, cells transfected with RNA derived from clone 2 ofpT7-IC-CEA-sig⁻ ; Lane 4, cells transfected with RNA derived from clone3 of pT7-IC-CEA-sig⁻ ; Lane 5, cells transfected with RNA derived fromclone 4 of pT7-IC-CEA-sig⁻ ; Lane 6, cells transfected with RNA derivedfrom clone 5 of pT7-IC-CEA-sig⁻ ; Lane 7, cells transfected with RNAderived from transcription of pT7-IC-Gag1; Lane 8, cells infected witheither poliovirus (FIG. 24A) or rV-CEA (FIG. 24B). The migration of themolecular mass markers is noted. The migration of 3CD (FIG. 24A) andglycosylated and unglycosylated forms of CEA (FIG. 24B) are also noted.

In contrast to the results with the CEA recombinant poliovirus nucleicacids encoding the signal sequence, the 3CD protein from cellstransfected with RNA derived from five individual clones ofpT7-IC-CEA-sig⁻ was detected. The levels of 3CD expression in thisexperiment were comparable to those of cells transfected with RNAderived from in vitro transcription of pT7-IC-Gag 1, which was knownfrom previous studies to be replication competent (Porter, D.C. et al.(1993)J. Virol. 67:3712-3719; FIG. 24A). To determine if the CEA proteinwas expressed in the transfected cells, the lysates were also incubatedwith the Col-1 antibody to immunoprecipitate CEA-related proteins (FIG.24B). Since the CEA protein should not be glycosylated, it was expectedthat the CEA product would be approximately 80 kDa in molecular mass. Ineach of the transfections with RNA derived the five independent clones,an 80-kDa protein was immunoprecipitated; this protein was not detectedin cells transfected with recombinant poliovirus nucleic acidscontaining the HIV-1 gag gene.

EXAMPLE 8 ENCAPSIDATION AND SERIAL PASSAGE OF RECOMBINANT POLIOVIRUSNUCLEIC ACID CONTAINING THE GENE FOR CARCINOEMBRYONIC ANTIGEN

To determine whether the recombinant poliovirus nucleic acids containingthe CEA sig⁻ gene could be encapsidated if provided the polioviruscapsid proteins, cells were infected first with VV-P 1, followed bytransfection with either the RNA derived pT7-IC-CEA-sig⁻ orPT7-IC-Gag 1. A mock transfection was also included as an additionalcontrol. At 24 h posttransfection, extracts of the cells were generatedby addition of detergents to the culture medium, and poliovirus-likeparticles were concentrated from the extracts by centrifugation througha 30% sucrose cushion. After resuspension, the concentrated material wasused to infect cells that had been infected previously with eitherwild-type vaccinia virus or VV-P1 (passage 1). This coinfection wasallowed to proceed overnight, after which extracts of the cells weregenerated by repeated freezing and thawing. The freeze-thaw extractswere clarified and used to repeat the coinfection procedure. Thisprocess was repeated for an additional nine serial passages to generatestocks of the encapsidated recombinant poliovirus nucleic acids. For theexperiment shown in FIGS. 25A-C, the lysates from Pass 10 material wereused to infect BSC-40 cells. At 6.5 hours postinfection, the cells werestarved for 30 min in methionine-cysteine-free DMEM, and then weremetabolically labeled for an additional 90 min. The cell lysates werethen analyzed by immunoprecipitation with either anti-3D^(pol) antibody(FIG. 25A) or antibody to the CEA protein (Col-1, FIG. 25B). The originsof the samples in the lanes for FIGS. 25A and 25B are as follows:Lane 1. cells that were infected with wild-type vaccinia virus and thenmock-transfected; Lane 2,cells that were infected with VV-P1 and thenmock-transfected; Lane 3, cells that were infected with wild-typevaccinia virus and then transfected with RNA derived from in vitrotranscription of pT7-IC-CEA-sig⁻ ; Lane 4, cells that were infected withVV-P1 and then transfected with RNA derived from pT7-IC-CEA-sig⁻ ; Lane5, cells that were infected with wild-type vaccinia virus and thentransfected with RNA derived from pT7-IC-CEA-sig⁻ (a second independentclone); Lane 6, cells were infected with VV-P1 and then transfected withRNA derived from pT7-IC-CEA-sig⁻ (a second independent clone); Lane 7,cells that were infected with wild-type vaccinia virus and thentransfected with RNA derived from in vitro transcription of pT7-IC-Gag1; Lane 8, cells that were infected with VV-P1 and then transfected withRNA derived from in vitro transcription of pT7-IC-Gag 1; Lane 9, cellsthat were infected with poliovirus (FIG. 25A) or recombinant vacciniavirus CEA (rV-CEA, FIG. 25B). The migration of the molecular massmarkers is noted. In FIG. 25A, the migration of 3CD protein is noted,whereas in FIG. 25B, the migrations of the glycosylated (gly) andnonglycosylated (sig⁻) forms of CEA are noted. Arrows note the positionof the anti-CEA immunoreactive proteins of larger molecular massobserved in cells infected with encapsidated poliovirus nucleic acidcontaining the signal-minus CEA gene. In FIG. 25C, cells were infectedwith a Pass 20 stock of encapsidated recombinant poliovirus nucleic acidcontaining the signal-minus CEA gene and then metabolically labeled with[³ H]leucine. The origins of the samples in the lanes for FIG. 25C areas follows: Lane 1 includes uninfected cells metabolically labeled,followed by immunoprecipitation with Col-1 antibody; Lane 2, cellsinfected with encapsidated recombinant poliovirus nucleic acidcontaining the signal-minus CEA gene, followed by immunoprecipitationwith Col-1 antibody. The molecular mass standards are noted as well asthe migration of glycosylated CEA (glyc.), nonglycosylated CEA (sig⁻),and breakdown product (asterisk).

No expression of 3CD proteins was detected upon infection of cells withthe sample originating from the mock-transfected cells and seriallypassaged 10 times with either wild-type vaccinia virus of VV-P1(FIG.25A). From analysis of 3CD expression, it was concluded that RNA derivedfrom transcription of pT7-IC-CEA-sig⁻ was encapsidated when passaged inthe presence of VV-P1, but not in the presence of wild-type vacciniavirus.

To determine if the CEA protein was expressed from the encapsidatedrecombinant poliovirus nucleic acids, the extracts from infected cellsthat had been metabolically labeled followed by immunoprecipitation withthe Col-1 antibody (FIG. 25B) were analyzed. Again, in samples frommock-transfected cells that had been subsequently passaged in thepresence of either wild-type vaccinia virus or VV-P1, no immunoreactiveprotein was detected. A protein of molecular mass 80 kDa wasimmunoprecipitated from cells infected with the extracts originatingfrom cells transfected with the RNA derived from pT7-IC-CEA sig⁻ whichhas been passaged in the presence of VV-P1, but not in the presence ofwild-type virus. As expected, no Col-1 immunoreactive material wasdetected in cells infected with the RNA derived from pT7-IC-Gag 1,although this RNA was encapsidated in cells in the presence of VV-P1(FIG. 25A).

Although the majority of the CEA protein immunoprecipitated from thecells infected with either stock of the encapsidated recombinantpoliovirus RNA was the 80-kDa protein corresponding to the expectedmolecular mass of unglycosylated CEA, it was noted there was a smallamount of protein immunoprecipitated corresponding to the molecular massfor the fully glycosylated CEA protein (180 kDa). To further explorethis result, a concentrated stock of the signal-minus CEA recombinantpoliovirus nucleic acid that had been passaged an additional 10 times(20 serial passages in all) and concentrated by pelleting through a 30%sucrose cushion prior to use in these experiments was used. Cells wereinfected with the encapsidated recombinant poliovirus nucleic acids,followed by metabolic radiolabeling for 1.5 h with [³ H]leucine sinceCEA contains more leucine amino acids than methionine or cysteine(Oikawa, S. et al. (1987)Biochim. Biophys. Acta. 142:511-518). Thisshould increase the sensitivity of detection of the higher molecularmass CEA proteins. Three proteins were immunoprecipitated using theCol-1 antibody from [³ H]leucine-labeled cells infected with the stockof the encapsidated recombinant poliovirus nucleic acid (FIG. 25C). Oneof these proteins corresponded to the unglycosylated protein of asmaller molecular mass of approximately 80 kDa, while a protein of asmaller molecular mass, corresponding to approximately 52 kDa, was alsoimmunoprecipitated. This protein is believed to represent a breakdownproduct of the CEA protein that was not detected previously because ofthe relatively few methionine or cysteine amino acids found in the CEAprotein. A third protein of approximately 180 kDa was alsoimmunoprecipitated, suggesting that glycosylated CEA protein might beproduced in cells infected with the encapsidated recombinant poliovirusnucleic acids at low levels.

EXAMPLE 9 PRODUCTION OF ANTI-POLIOVIRUS AND ANTI-CARCINOEMBRYONICANTIGEN ANTIBODIES IN MICE IMMUNIZED WITH ENCAPSIDATED RECOMBINANTPOLIOVIRUS NUCLEIC ACID CONTAINING THE GENE FOR CARCINOEMBRYONIC ANTIGEN

To evaluate the immunogenicity of the encapsidated recombinantpoliovirus nucleic acids which express the CEA protein, transgenic micethat express the receptor for poliovirus and are susceptible toinfection with poliovirus were used (Ren, R. et al. (1990)Cell63:353-362). The mice were bred in a germ-free environment until use inthe experiments. The four mice used in the experiment were bled prior toi.m. immunization with approximately 10⁴ infectious units of theencapsidated recombinant poliovirus nucleic acid which expresses CEA.The serum samples from the mice at each of the pre- and postimmune timepoints were pooled and assayed using a solid-phase ELISA with wholepoliovirus or recombinant CEA expressed in E. coli as the coatingsolution. The results are presented as absorbance 414-nm values at afixed dilution and as end point titration values for anti-CEA (FIG. 26A)an antipoliovirus (FIG. 26B). By 28 days after the second boosterimmunization, a pronounced CEA-specific antibody response was detectedas measured by the ELISA assay.

The end point titer had increased from 1:25 (preimmune) to 1:6400 (FIG.26A). A similar increase was observed in the antipoliovirus in the serumsamples (FIG. 26B). As a control, no increase in anti-CEA antibodies inthe sera from mice immunized with the recombinant poliovirus nucleicacid expressing HIV-1 Gag was found. Taken together, these resultsdemonstrate that the recombinant poliovirus nucleic acids infect cells,presumably the muscle myofibers at the site of injection, and expresssufficient amounts of CEA to stimulate an anti-CEA antibody response.

The construction and characterization of RNA recombinant poliovirusnucleic acids which express the CEA protein when infected is describedherein. A recombinant poliovirus nucleic acid encoding the signal-minusCEA protein was replication competent and expressed nonglycosylated CEAprotein when transfected into cells. Using the methods of encapsidatingrecombinant poliovirus nucleic acids described herein, stocks ofencapsidated recombinant poliovirus nucleic acids containing thesignal-minus CEA gene were generated.

The use of encapsidated poliovirus recombinant poliovirus nucleic acidsas a vaccine vehicle has several distinguishing features: (a) this isone of the few vector systems based entirely on an RNA virus. Sincepoliovirus replication does not involve DNA intermediates, in contrastto retroviruses, the possibility of recombination in the host cell DNAis virtually eliminated; (b) infection of cells with encapsidatedrecombinant poliovirus nucleic acids results in an amplification of therecombinant poliovirus nucleic acid RNA and preferential expression ofthe foreign gene over cellular gene products since poliovirus hasevolved mechanisms to promote the synthesis of its own viral proteins(Ehrenfeld, E. et al. (1982)Cell 28:435-436); and (c) the encapsidatedpoliovirus recombinant poliovirus nucleic acids are noninfectiousbecause they do not encode the viral P1 capsid proteins. The recombinantpoliovirus nucleic acid requires capsid proteins to be propagated andtransmitted from cell to cell. Infection of cells or an animal with theencapsidated recombinant poliovirus nucleic acids alone then results ina single round of infection without a chance for further spread. Becauseof this feature, the encapsidated recombinant poliovirus nucleic acidscan be exploited to deliver nucleic acids to cells without risk of viralspread.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All referenced patents and publications are hereby incorporated byreference in their entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 23                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TATTAGTAGA TCTG              - #                  - #                      - #     14                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TACAGATGTA CTAA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 845 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 20..845                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - ACACAGCAAT CAGGTCAGC CAA AAT TAC CCT ATA GTG CAG - # AAC ATC CAG        GGG      52                                                                                       - #   Gln Asn Tyr Pro Ile Val Gln Asn Ile - # Gln Gly                        - #     1             - #  5                - #  10          - - CAA ATG GTA CAT CAG GCC ATA TCA CCT AGA AC - #T TTA AAT GCA TGG GTA          100                                                                       Gln Met Val His Gln Ala Ile Ser Pro Arg Th - #r Leu Asn Ala Trp Val                        15     - #             20     - #             25                  - - AAA GTA GTA GAA GAG AAG GCT TTC AGC CCA GA - #A GTG ATA CCC ATG TTT          148                                                                       Lys Val Val Glu Glu Lys Ala Phe Ser Pro Gl - #u Val Ile Pro Met Phe                    30         - #         35         - #         40                      - - TCA GCA TTA TCA GAA GGA GCC ACC CCA CAA GA - #T TTA AAC ACC ATG CTA          196                                                                       Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln As - #p Leu Asn Thr Met Leu                45             - #     50             - #     55                          - - AAC ACA GTG GGG GGA CAT CAA GCA GCC ATG CA - #A ATG TTA AAA GAG ACC          244                                                                       Asn Thr Val Gly Gly His Gln Ala Ala Met Gl - #n Met Leu Lys Glu Thr            60                 - # 65                 - # 70                 - # 75       - - ATC AAT GAG GAA GCT GCA GAA TGG GAT AGA GT - #G CAT CCA GTG CAT GCA          292                                                                       Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Va - #l His Pro Val His Ala                            80 - #                 85 - #                 90              - - GGG CCT ATT GCA CCA GGC CAG ATG AGA GAA CC - #A AGG GGA AGT GAC ATA          340                                                                       Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pr - #o Arg Gly Ser Asp Ile                        95     - #            100     - #            105                  - - GCA GGA ACT ACT AGT ACC CTT CAG GAA CAA AT - #A GGA TGG ATG ACA AAT          388                                                                       Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Il - #e Gly Trp Met Thr Asn                   110          - #       115          - #       120                      - - AAT CCA CCT ATC CCA GTA GGA GAA ATT TAT AA - #A AGA TGG ATA ATC CTG          436                                                                       Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Ly - #s Arg Trp Ile Ile Leu               125              - #   130              - #   135                          - - GGA TTA AAT AAA ATA GTA AGA ATG TAT AGC CC - #T ACC AGC ATT CTG GAC          484                                                                       Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pr - #o Thr Ser Ile Leu Asp           140                 1 - #45                 1 - #50                 1 -      #55                                                                              - - ATA AGA CAA GGA CCA AAG GAA CCC TTT AGA GA - #C TAT GTA GAC CGG        TTC      532                                                                    Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg As - #p Tyr Val Asp Arg Phe                          160  - #               165  - #               170              - - TAT AAA ACT CTA AGA GCC GAG CAA GCT TCA CA - #G GAG GTA AAA AAT TGG          580                                                                       Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser Gl - #n Glu Val Lys Asn Trp                       175      - #           180      - #           185                  - - ATG ACA GAA ACC TTG TTG GTC CAA AAT GCG AA - #C CCA GAT TGT AAG ACT          628                                                                       Met Thr Glu Thr Leu Leu Val Gln Asn Ala As - #n Pro Asp Cys Lys Thr                   190          - #       195          - #       200                      - - ATT TTA AAA GCA TTG GGA CCA GCG GCT ACA CT - #A GAA GAA ATG ATG ACA          676                                                                       Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Le - #u Glu Glu Met Met Thr               205              - #   210              - #   215                          - - GCA TGT CAG GGA GTA GGA GGA CCC GGC CAT AA - #G GCA AGA GTT TTG GCT          724                                                                       Ala Cys Gln Gly Val Gly Gly Pro Gly His Ly - #s Ala Arg Val Leu Ala           220                 2 - #25                 2 - #30                 2 -      #35                                                                              - - GAA GCA ATG AGC CAA GTA ACA AAT TCA GCT AC - #C ATA ATG ATG CAG        AGA      772                                                                    Glu Ala Met Ser Gln Val Thr Asn Ser Ala Th - #r Ile Met Met Gln Arg                          240  - #               245  - #               250              - - GGC AAT TTT AGG AAC CAA AGA AAG ATT GTT AA - #G TGT TTC AAT TGT GGC          820                                                                       Gly Asn Phe Arg Asn Gln Arg Lys Ile Val Ly - #s Cys Phe Asn Cys Gly                       255      - #           260      - #           265                  - - AAA GAA GGG CAC ACA GCC AGA AAG T    - #                  - #                  845                                                                     Lys Glu Gly His Thr Ala Arg Lys                                                       270          - #       275                                             - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 275 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Gln Asn Tyr Pro Ile Val Gln Asn Ile Gln Gl - #y Gln Met Val His Gln        1               5 - #                 10 - #                 15              - - Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Va - #l Lys Val Val Glu Glu                   20     - #             25     - #             30                  - - Lys Ala Phe Ser Pro Glu Val Ile Pro Met Ph - #e Ser Ala Leu Ser Glu               35         - #         40         - #         45                      - - Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Le - #u Asn Thr Val Gly Gly           50             - #     55             - #     60                          - - His Gln Ala Ala Met Gln Met Leu Lys Glu Th - #r Ile Asn Glu Glu Ala       65                 - # 70                 - # 75                 - # 80       - - Ala Glu Trp Asp Arg Val His Pro Val His Al - #a Gly Pro Ile Ala Pro                       85 - #                 90 - #                 95              - - Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Il - #e Ala Gly Thr Thr Ser                  100      - #           105      - #           110                  - - Thr Leu Gln Glu Gln Ile Gly Trp Met Thr As - #n Asn Pro Pro Ile Pro              115          - #       120          - #       125                      - - Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Le - #u Gly Leu Asn Lys Ile          130              - #   135              - #   140                          - - Val Arg Met Tyr Ser Pro Thr Ser Ile Leu As - #p Ile Arg Gln Gly Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Ph - #e Tyr Lys Thr Leu        Arg                                                                                             165  - #               170  - #               175             - - Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Tr - #p Met Thr Glu Thr Leu                  180      - #           185      - #           190                  - - Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Th - #r Ile Leu Lys Ala Leu              195          - #       200          - #       205                      - - Gly Pro Ala Ala Thr Leu Glu Glu Met Met Th - #r Ala Cys Gln Gly Val          210              - #   215              - #   220                          - - Gly Gly Pro Gly His Lys Ala Arg Val Leu Al - #a Glu Ala Met Ser Gln      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Thr Asn Ser Ala Thr Ile Met Met Gln Ar - #g Gly Asn Phe Arg        Asn                                                                                             245  - #               250  - #               255             - - Gln Arg Lys Ile Val Lys Cys Phe Asn Cys Gl - #y Lys Glu Gly His Thr                  260      - #           265      - #           270                  - - Ala Arg Lys                                                                      275                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 948 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..946                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AAC CAA TGG CCA TTG ACA GAA GAA AAA ATA AA - #A GCA TTA GTA GAA ATT           48                                                                           Gln Trp Pro Leu Thr Glu Glu Lys - #Ile Lys Ala Leu Val Glu Ile                  1            - #   5               - #   10               - #   15       - - TGT ACA GAG ATG GAA AAG GAA GGG AAA ATT TC - #A AAA ATT GGG CCT GAA           96                                                                       Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Se - #r Lys Ile Gly Pro Glu                            20 - #                 25 - #                 30              - - AAT CCA TAC AAT ACT CCA GTA TTT GCC ATA AA - #G AAA AAA GAC AGT ACT          144                                                                       Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile Ly - #s Lys Lys Asp Ser Thr                        35     - #             40     - #             45                  - - AAA TGG AGA AAA TTA GTA GAT TTC AGA GAA CT - #T AAT AAG AGA ACT CAA          192                                                                       Lys Trp Arg Lys Leu Val Asp Phe Arg Glu Le - #u Asn Lys Arg Thr Gln                    50         - #         55         - #         60                      - - GAC TTC TGG GAA GTT CAA TTA GGA ATA CCA CA - #T CCC GCA GGG TTA AAA          240                                                                       Asp Phe Trp Glu Val Gln Leu Gly Ile Pro Hi - #s Pro Ala Gly Leu Lys                65             - #     70             - #     75                          - - AAG AAA AAA TCA GTA ACA GTA CTG GAT GTG GG - #T GAT GCA TAT TTT TCA          288                                                                       Lys Lys Lys Ser Val Thr Val Leu Asp Val Gl - #y Asp Ala Tyr Phe Ser            80                 - # 85                 - # 90                 - # 95       - - GTT CCC TTA GAT GAA GAC TTC AGG AAG TAT AC - #T GCA TTT ACC ATA CCT          336                                                                       Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr Th - #r Ala Phe Thr Ile Pro                           100  - #               105  - #               110              - - AGT ATA AAC AAT GAG ACA CCA GGG ATT AGA TA - #T CAG TAC AAT GTG CTT          384                                                                       Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg Ty - #r Gln Tyr Asn Val Leu                       115      - #           120      - #           125                  - - CCA CAG GGA TGG AAA GGA TCA CCA GCA ATA TT - #C CAA AGT AGC ATG ACA          432                                                                       Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile Ph - #e Gln Ser Ser Met Thr                   130          - #       135          - #       140                      - - AAA ATC TTA GAG CCT TTT AGA AAA CAA AAT CC - #A GAC ATA GTT ATC TAT          480                                                                       Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pr - #o Asp Ile Val Ile Tyr               145              - #   150              - #   155                          - - CAA TAC ATG GAT GAT TTG TAT GTA GGA TCT GA - #C TTA GAA ATA GGG CAG          528                                                                       Gln Tyr Met Asp Asp Leu Tyr Val Gly Ser As - #p Leu Glu Ile Gly Gln           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - CAT AGA ACA AAA ATA GAG GAG CTG AGA CAA CA - #T CTG TTG AGG TGG        GGA      576                                                                    His Arg Thr Lys Ile Glu Glu Leu Arg Gln Hi - #s Leu Leu Arg Trp Gly                          180  - #               185  - #               190              - - CTT ACC ACA CCA GAC AAA AAA CAT CAG AAA GA - #A CCT CCA TTC CTT TGG          624                                                                       Leu Thr Thr Pro Asp Lys Lys His Gln Lys Gl - #u Pro Pro Phe Leu Trp                       195      - #           200      - #           205                  - - ATG GGT TAT GAA CTC CAT CCT GAT AAA TGG AC - #A GTA CAG CCT ATA GTG          672                                                                       Met Gly Tyr Glu Leu His Pro Asp Lys Trp Th - #r Val Gln Pro Ile Val                   210          - #       215          - #       220                      - - CTG CCA GAA AAA GAC AGC TGG ACT GTC AAT GA - #C ATA CAG AAG TTA GTG          720                                                                       Leu Pro Glu Lys Asp Ser Trp Thr Val Asn As - #p Ile Gln Lys Leu Val               225              - #   230              - #   235                          - - GGG AAA TTG AAT TGG GCA AGT CAG ATT TAC CC - #A GGG ATT AAA GTA AGG          768                                                                       Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Pr - #o Gly Ile Lys Val Arg           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - CAA TTA TGT AAA CTC CTT AGA GGA ACC AAA GC - #A CTA ACA GAA GTA        ATA      816                                                                    Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys Al - #a Leu Thr Glu Val Ile                          260  - #               265  - #               270              - - CCA CTA ACA GAA GAA GCA GAG CTA GAA CTG GC - #A GAA AAC AGA GAG ATT          864                                                                       Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu Al - #a Glu Asn Arg Glu Ile                       275      - #           280      - #           285                  - - CTA AAA GAA CCA GTA CAT GGA GTG TAT TAT GA - #C CCA TCA AAA GAC TTA          912                                                                       Leu Lys Glu Pro Val His Gly Val Tyr Tyr As - #p Pro Ser Lys Asp Leu                   290          - #       295          - #       300                      - - ATA GCA GAA ATA CAG AAG CAG GGG CAA GGC CT - #CGAG                      - #      948                                                                    Ile Ala Glu Ile Gln Lys Gln Gly Gln Gly                                           305              - #   310                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 314 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Al - #a Leu Val Glu Ile        Cys                                                                               1               5 - #                 10 - #                 15             - - Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Ly - #s Ile Gly Pro Glu Asn                   20     - #             25     - #             30                  - - Pro Tyr Asn Thr Pro Val Phe Ala Ile Lys Ly - #s Lys Asp Ser Thr Lys               35         - #         40         - #         45                      - - Trp Arg Lys Leu Val Asp Phe Arg Glu Leu As - #n Lys Arg Thr Gln Asp           50             - #     55             - #     60                          - - Phe Trp Glu Val Gln Leu Gly Ile Pro His Pr - #o Ala Gly Leu Lys Lys       65                 - # 70                 - # 75                 - # 80       - - Lys Lys Ser Val Thr Val Leu Asp Val Gly As - #p Ala Tyr Phe Ser Val                       85 - #                 90 - #                 95              - - Pro Leu Asp Glu Asp Phe Arg Lys Tyr Thr Al - #a Phe Thr Ile Pro Ser                  100      - #           105      - #           110                  - - Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gl - #n Tyr Asn Val Leu Pro              115          - #       120          - #       125                      - - Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gl - #n Ser Ser Met Thr Lys          130              - #   135              - #   140                          - - Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro As - #p Ile Val Ile Tyr Gln      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp Le - #u Glu Ile Gly Gln        His                                                                                             165  - #               170  - #               175             - - Arg Thr Lys Ile Glu Glu Leu Arg Gln His Le - #u Leu Arg Trp Gly Leu                  180      - #           185      - #           190                  - - Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pr - #o Pro Phe Leu Trp Met              195          - #       200          - #       205                      - - Gly Tyr Glu Leu His Pro Asp Lys Trp Thr Va - #l Gln Pro Ile Val Leu          210              - #   215              - #   220                          - - Pro Glu Lys Asp Ser Trp Thr Val Asn Asp Il - #e Gln Lys Leu Val Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Lys Leu Asn Trp Ala Ser Gln Ile Tyr Pro Gl - #y Ile Lys Val Arg        Gln                                                                                             245  - #               250  - #               255             - - Leu Cys Lys Leu Leu Arg Gly Thr Lys Ala Le - #u Thr Glu Val Ile Pro                  260      - #           265      - #           270                  - - Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Gl - #u Asn Arg Glu Ile Leu              275          - #       280          - #       285                      - - Lys Glu Pro Val His Gly Val Tyr Tyr Asp Pr - #o Ser Lys Asp Leu Ile          290              - #   295              - #   300                          - - Ala Glu Ile Gln Lys Gln Gly Gln Gly Leu                                  305                 3 - #10                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1568 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..1565                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GGGGCC TGT CCA AAG GTA TCC TTT GAG CCA ATT - #CCC ATA CAT TAT TGT            48                                                                               Cys Pro Lys Val Ser Phe Gl - #u Pro Ile Pro Ile His Tyr Cys                     1         - #      5            - #      10                           - - GCC CCG GCT GGT TTT GCG ATT CTA AAA TGT AA - #T AAT AAG ACG TTC AAT           96                                                                       Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys As - #n Asn Lys Thr Phe Asn            15                 - # 20                 - # 25                 - # 30       - - GGA ACA GGA CCA TGT ACA AAT GTC AGC ACA GT - #A CAA TGT ACA CAT GGA          144                                                                       Gly Thr Gly Pro Cys Thr Asn Val Ser Thr Va - #l Gln Cys Thr His Gly                            35 - #                 40 - #                 45              - - ATT AGG CCA GTA GTA TCA ACT CAA CTG CTG TT - #A AAT GGC AGT CTA GCA          192                                                                       Ile Arg Pro Val Val Ser Thr Gln Leu Leu Le - #u Asn Gly Ser Leu Ala                        50     - #             55     - #             60                  - - GAA GAA GAG GTA GTA ATT AGA TCT GTC AAT TT - #C ACG GAC AAT GCT AAA          240                                                                       Glu Glu Glu Val Val Ile Arg Ser Val Asn Ph - #e Thr Asp Asn Ala Lys                    65         - #         70         - #         75                      - - ACC ATA ATA GTA CAG CTG AAC ACA TCT GTA GA - #A ATT AAT TGT ACA AGA          288                                                                       Thr Ile Ile Val Gln Leu Asn Thr Ser Val Gl - #u Ile Asn Cys Thr Arg                80             - #     85             - #     90                          - - CCC AAC AAC AAT ACA AGA AAA AGA ATC CGT AT - #C CAG AGA GGA CCA GGG          336                                                                       Pro Asn Asn Asn Thr Arg Lys Arg Ile Arg Il - #e Gln Arg Gly Pro Gly            95                 - #100                 - #105                 - #110       - - AGA GCA TTT GTT ACA ATA GGA AAA ATA GGA AA - #T ATG AGA CAA GCA CAT          384                                                                       Arg Ala Phe Val Thr Ile Gly Lys Ile Gly As - #n Met Arg Gln Ala His                           115  - #               120  - #               125              - - TGT AAC ATT AGT AGA GCA AAA TGG AAT AAC AC - #T TTA AAA CAG ATA GAT          432                                                                       Cys Asn Ile Ser Arg Ala Lys Trp Asn Asn Th - #r Leu Lys Gln Ile Asp                       130      - #           135      - #           140                  - - AGC AAA TTA AGA GAA CAA TTC GGA AAT AAT AA - #A ACA ATA ATC TTT AAG          480                                                                       Ser Lys Leu Arg Glu Gln Phe Gly Asn Asn Ly - #s Thr Ile Ile Phe Lys                   145          - #       150          - #       155                      - - CAA TCC TCA GGA GGG GAC CCA GAA ATT GTA AC - #G CAC AGT TTT AAT TGT          528                                                                       Gln Ser Ser Gly Gly Asp Pro Glu Ile Val Th - #r His Ser Phe Asn Cys               160              - #   165              - #   170                          - - GGA GGG GAA TTT TTC TAC TGT AAT TCA ACA CA - #A CTG TTT AAT AGT ACT          576                                                                       Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gl - #n Leu Phe Asn Ser Thr           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - TGG TTT AAT AGT ACT TGG AGT ACT GAA GGG TC - #A AAT AAC ACT GAA        GGA      624                                                                    Trp Phe Asn Ser Thr Trp Ser Thr Glu Gly Se - #r Asn Asn Thr Glu Gly                          195  - #               200  - #               205              - - AGT GAC ACA ATC ACC CTC CCA TGC AGA ATA AA - #A CAA ATT ATA AAC ATG          672                                                                       Ser Asp Thr Ile Thr Leu Pro Cys Arg Ile Ly - #s Gln Ile Ile Asn Met                       210      - #           215      - #           220                  - - TGG CAG AAA GTA GGA AAA GCA ATG TAT GCC CC - #T CCC ATC AGT GGA CAA          720                                                                       Trp Gln Lys Val Gly Lys Ala Met Tyr Ala Pr - #o Pro Ile Ser Gly Gln                   225          - #       230          - #       235                      - - ATT AGA TGT TCA TCA AAT ATT ACA GGG CTG CT - #A TTA ACA AGA GAT GGT          768                                                                       Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Le - #u Leu Thr Arg Asp Gly               240              - #   245              - #   250                          - - GGT AAT AGC AAC AAT GAG TCC GAG ATC TTC AG - #A CTT GGA GGA GGA GAT          816                                                                       Gly Asn Ser Asn Asn Glu Ser Glu Ile Phe Ar - #g Leu Gly Gly Gly Asp           255                 2 - #60                 2 - #65                 2 -      #70                                                                              - - ATG AGG GAC AAT TGG AGA AGT GAA TTA TAT AA - #A TAT AAA GTA GTA        AAA      864                                                                    Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Ly - #s Tyr Lys Val Val Lys                          275  - #               280  - #               285              - - ATT GAA CCA TTA GGA GTA GCA CCC ACC AAG GC - #A AAG AGA AGA GTG GTG          912                                                                       Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Al - #a Lys Arg Arg Val Val                       290      - #           295      - #           300                  - - CAG AGA GAA AAA AGA GCA GTG GGA ATA GGA GC - #T TTG TTC CTT GGG TTC          960                                                                       Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Al - #a Leu Phe Leu Gly Phe                   305          - #       310          - #       315                      - - TTG GGA GCA GCA GGA AGC ACT ATG GGC GCA GC - #C TCA ATG ACG CTG ACG         1008                                                                       Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Al - #a Ser Met Thr Leu Thr               320              - #   325              - #   330                          - - GTA CAG GCC AGA CAA TTA TTG TCT GGT ATA GT - #G CAG CAG CAG AAC AAT         1056                                                                       Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Va - #l Gln Gln Gln Asn Asn           335                 3 - #40                 3 - #45                 3 -      #50                                                                              - - TTG CTG AGG GCT ATT GAG GCG CAA CAG CAT CT - #G TTG CAA CTC ACA        GTC     1104                                                                    Leu Leu Arg Ala Ile Glu Ala Gln Gln His Le - #u Leu Gln Leu Thr Val                          355  - #               360  - #               365              - - TGG GGC ATC AAG CAG CTC CAA GCA AGA ATC CT - #A GCT GTG GAA AGA TAC         1152                                                                       Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Le - #u Ala Val Glu Arg Tyr                       370      - #           375      - #           380                  - - CTA AAG GAT CAA CAG CTC CTA GGG ATT TGG GG - #T TGC TCT GGA AAA CTC         1200                                                                       Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gl - #y Cys Ser Gly Lys Leu                   385          - #       390          - #       395                      - - ATT TGC ACC ACT GCT GTG CCT TGG AAT GCT AG - #T TGG AGT AAT AAA TCT         1248                                                                       Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Se - #r Trp Ser Asn Lys Ser               400              - #   405              - #   410                          - - CTG GAA CAG ATC TGG AAT CAC ACG ACC TGG AT - #G GAG TGG GAC AGA GAA         1296                                                                       Leu Glu Gln Ile Trp Asn His Thr Thr Trp Me - #t Glu Trp Asp Arg Glu           415                 4 - #20                 4 - #25                 4 -      #30                                                                              - - ATT AAC AAT TAC ACA AGC TTA ATA CAC TCC TT - #A ATT GAA GAA TCG        CAA     1344                                                                    Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Le - #u Ile Glu Glu Ser Gln                          435  - #               440  - #               445              - - AAC CAG CAA GAA AAG AAT GAA CAA GAA TTA TT - #G GAA TTA GAT AAA TGG         1392                                                                       Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Le - #u Glu Leu Asp Lys Trp                       450      - #           455      - #           460                  - - GCA AGT TTG TGG AAT TGG TTT AAC ATA ACA AA - #T TGG CTG TGG TAT ATA         1440                                                                       Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr As - #n Trp Leu Trp Tyr Ile                   465          - #       470          - #       475                      - - AAA TTA TTC ATA ATG ATA GTA GGA GGC TTG GT - #A GGT TTA AGA ATA GTT         1488                                                                       Lys Leu Phe Ile Met Ile Val Gly Gly Leu Va - #l Gly Leu Arg Ile Val               480              - #   485              - #   490                          - - TTT GCT GTA CTT TCT ATA GTG AAT AGA GTT AG - #G CAG GGA TAT TCA CCA         1536                                                                       Phe Ala Val Leu Ser Ile Val Asn Arg Val Ar - #g Gln Gly Tyr Ser Pro           495                 5 - #00                 5 - #05                 5 -      #10                                                                              - - TTA TCG TTT CAG ACC CAC CTC CCA ATC TCGAG - #                  - #            1568                                                                    Leu Ser Phe Gln Thr His Leu Pro Ile                                                           515                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 519 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Il - #e His Tyr Cys Ala Pro        1               5 - #                 10 - #                 15              - - Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn Ly - #s Thr Phe Asn Gly Thr                   20     - #             25     - #             30                  - - Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cy - #s Thr His Gly Ile Arg               35         - #         40         - #         45                      - - Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gl - #y Ser Leu Ala Glu Glu           50             - #     55             - #     60                          - - Glu Val Val Ile Arg Ser Val Asn Phe Thr As - #p Asn Ala Lys Thr Ile       65                 - # 70                 - # 75                 - # 80       - - Ile Val Gln Leu Asn Thr Ser Val Glu Ile As - #n Cys Thr Arg Pro Asn                       85 - #                 90 - #                 95              - - Asn Asn Thr Arg Lys Arg Ile Arg Ile Gln Ar - #g Gly Pro Gly Arg Ala                  100      - #           105      - #           110                  - - Phe Val Thr Ile Gly Lys Ile Gly Asn Met Ar - #g Gln Ala His Cys Asn              115          - #       120          - #       125                      - - Ile Ser Arg Ala Lys Trp Asn Asn Thr Leu Ly - #s Gln Ile Asp Ser Lys          130              - #   135              - #   140                          - - Leu Arg Glu Gln Phe Gly Asn Asn Lys Thr Il - #e Ile Phe Lys Gln Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Gly Gly Asp Pro Glu Ile Val Thr His Se - #r Phe Asn Cys Gly        Gly                                                                                             165  - #               170  - #               175             - - Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Ph - #e Asn Ser Thr Trp Phe                  180      - #           185      - #           190                  - - Asn Ser Thr Trp Ser Thr Glu Gly Ser Asn As - #n Thr Glu Gly Ser Asp              195          - #       200          - #       205                      - - Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln          210              - #   215              - #   220                          - - Lys Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Ser Gly Gln Ile Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly        Asn                                                                                             245  - #               250  - #               255             - - Ser Asn Asn Glu Ser Glu Ile Phe Arg Leu Gl - #y Gly Gly Asp Met Arg                  260      - #           265      - #           270                  - - Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Ly - #s Val Val Lys Ile Glu              275          - #       280          - #       285                      - - Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Ar - #g Arg Val Val Gln Arg          290              - #   295              - #   300                          - - Glu Lys Arg Ala Val Gly Ile Gly Ala Leu Ph - #e Leu Gly Phe Leu Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Me - #t Thr Leu Thr Val        Gln                                                                                             325  - #               330  - #               335             - - Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gl - #n Gln Asn Asn Leu Leu                  340      - #           345      - #           350                  - - Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gl - #n Leu Thr Val Trp Gly              355          - #       360          - #       365                      - - Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Va - #l Glu Arg Tyr Leu Lys          370              - #   375              - #   380                          - - Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Se - #r Gly Lys Leu Ile Cys      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Se - #r Asn Lys Ser Leu        Glu                                                                                             405  - #               410  - #               415             - - Gln Ile Trp Asn His Thr Thr Trp Met Glu Tr - #p Asp Arg Glu Ile Asn                  420      - #           425      - #           430                  - - Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Gl - #u Glu Ser Gln Asn Gln              435          - #       440          - #       445                      - - Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Le - #u Asp Lys Trp Ala Ser          450              - #   455              - #   460                          - - Leu Trp Asn Trp Phe Asn Ile Thr Asn Trp Le - #u Trp Tyr Ile Lys Leu      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Phe Ile Met Ile Val Gly Gly Leu Val Gly Le - #u Arg Ile Val Phe        Ala                                                                                             485  - #               490  - #               495             - - Val Leu Ser Ile Val Asn Arg Val Arg Gln Gl - #y Tyr Ser Pro Leu Ser                  500      - #           505      - #           510                  - - Phe Gln Thr His Leu Pro Ile                                                      515                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CACCCCTCTC CTACGTAACC AAGGATC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GTACTGGTCA CCATATTGGT CAAC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GGAGAGAGAT GGGAGCTCGA GCGTC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GCCCCCCTAT ACGTATTGTG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CCAGTGAATT CCTAATACGA CTCACTATAG GTTAAAACAG C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CTCTATCCTG AGCTCCATAT GTGTCGAGCA GTTTTTGGTT TAGCATTG  - #                    48                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Thr Lys Asp Leu Thr Thr Tyr Gly                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2220 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2203                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CGA CCA GCA GAC CAG ACA GTC ACA GCA GCC TT - #G ACA AAA CGT TCC TGG           48                                                                       Arg Pro Ala Asp Gln Thr Val Thr Ala Ala Le - #u Thr Lys Arg Ser Trp             1               5 - #                 10 - #                 15              - - AAC TCA AGC ACT TCT CCA CAG AGG AGG ACA GA - #G CAG ACA GCA GAG ACC           96                                                                       Asn Ser Ser Thr Ser Pro Gln Arg Arg Thr Gl - #u Gln Thr Ala Glu Thr                        20     - #             25     - #             30                  - - ATG GAG TCT CCC TCG GCC CCT CCC CAC AGA TG - #G TGC ATC CCC TGG CAG          144                                                                       Met Glu Ser Pro Ser Ala Pro Pro His Arg Tr - #p Cys Ile Pro Trp Gln                    35         - #         40         - #         45                      - - AGG CTC CTG CTC ACA GCC TCA CTT CTA ACC TT - #C TGG AAC CCG CCC ACC          192                                                                       Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Ph - #e Trp Asn Pro Pro Thr                50             - #     55             - #     60                          - - ACT GCC AAG CTC ACT ATT GAA TCC ACG CCG TT - #C AAT GTC GCA GAG GGG          240                                                                       Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Ph - #e Asn Val Ala Glu Gly            65                 - # 70                 - # 75                 - # 80       - - AAG GAG GTG CTT CTA CTT GTC CAC AAT CTG CC - #C CAG CAT CTT TTT GGC          288                                                                       Lys Glu Val Leu Leu Leu Val His Asn Leu Pr - #o Gln His Leu Phe Gly                            85 - #                 90 - #                 95              - - TAC AGC TGG TAC AAA GGT GAA AGA GTG GAT GG - #C AAC CGT CAA ATT ATA          336                                                                       Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gl - #y Asn Arg Gln Ile Ile                       100      - #           105      - #           110                  - - GGA TAT GTA ATA GGA ACT CAA CAA GCT ACC CC - #A GGG CCC GCA TAC AGT          384                                                                       Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pr - #o Gly Pro Ala Tyr Ser                   115          - #       120          - #       125                      - - GGT CGA GAG ATA ATA TAC CCC AAT GCA TCC CT - #G CTG ATC CAG AAC ATC          432                                                                       Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Le - #u Leu Ile Gln Asn Ile               130              - #   135              - #   140                          - - ATC CAG AAT GAC ACA GGA TTC TAC ACC CTA CA - #C GTC ATA AAG TCA GAT          480                                                                       Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu Hi - #s Val Ile Lys Ser Asp           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - CTT GTG AAT GAA GAA GCA ACT GGC CAG TTC CG - #G GTA TAC CCG GAG        CTG      528                                                                    Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Ar - #g Val Tyr Pro Glu Leu                          165  - #               170  - #               175              - - CCC AAG CCC TCC ATC TCC AGC AAC AAC TCC AA - #A CCC GTG GAG GAC AAG          576                                                                       Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Ly - #s Pro Val Glu Asp Lys                       180      - #           185      - #           190                  - - GAT GCT GTG GCC TTC ACC TGT GAA CCT GAG AC - #T CAG GAC GCA ACC TAC          624                                                                       Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Th - #r Gln Asp Ala Thr Tyr                   195          - #       200          - #       205                      - - CTG TGG TGG GTA AAC AAT CAG AGC CTC CCG GT - #C AGT CCC AGG CTG CAG          672                                                                       Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Va - #l Ser Pro Arg Leu Gln               210              - #   215              - #   220                          - - CTG TCC AAT GGC AAC AGG ACC CTC ACT CTA TT - #C AAT GTC ACA AGA AAT          720                                                                       Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Ph - #e Asn Val Thr Arg Asn           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GAC ACA GCA AGC TAC AAA TGT GAA ACC CAG AA - #C CCA GTG AGT GCC        AGG      768                                                                    Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln As - #n Pro Val Ser Ala Arg                          245  - #               250  - #               255              - - CGC AGT GAT TCA GTC ATC CTG AAT GTC CTC TA - #T GGC CCG GAT GCC CCC          816                                                                       Arg Ser Asp Ser Val Ile Leu Asn Val Leu Ty - #r Gly Pro Asp Ala Pro                       260      - #           265      - #           270                  - - ACC ATT TCC CCT CTA AAC ACA TCT TAC AGA TC - #A GGG GAA AAT CTG AAC          864                                                                       Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Se - #r Gly Glu Asn Leu Asn                   275          - #       280          - #       285                      - - CTC TCC TGC CAT GCA GCC TCT AAC CCA CCT GC - #A CAG TAC TCT TGG TTT          912                                                                       Leu Ser Cys His Ala Ala Ser Asn Pro Pro Al - #a Gln Tyr Ser Trp Phe               290              - #   295              - #   300                          - - GTC AAT GGG ACT TTC CAG CAA TCC ACC CAA GA - #G CTC TTT ATC CCC AAC          960                                                                       Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Gl - #u Leu Phe Ile Pro Asn           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ATC ACT GTG AAT AAT AGT GGA TCC TAT ACG TG - #C CAA GCC CAT AAC        TCA     1008                                                                    Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cy - #s Gln Ala His Asn Ser                          325  - #               330  - #               335              - - GAC ACT GGC CTC AAT AGG ACC ACA GTC ACG AC - #G ATC ACA GTC TAT GCA         1056                                                                       Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Th - #r Ile Thr Val Tyr Ala                       340      - #           345      - #           350                  - - GAG CCA CCC AAA CCC TTC ATC ACC AGC AAC AA - #C TCC AAC CCC GTG GAG         1104                                                                       Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn As - #n Ser Asn Pro Val Glu                   355          - #       360          - #       365                      - - GAT GAG GAT GCT GTA GCC TTA ACC TGT GAA CC - #T GAG ATT CAG AAC ACA         1152                                                                       Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pr - #o Glu Ile Gln Asn Thr               370              - #   375              - #   380                          - - ACC TAC CTG TGG TGG GTA AAT AAT CAG AGC CT - #C CCG GTC AGT CCC AGG         1200                                                                       Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Le - #u Pro Val Ser Pro Arg           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - CTG CAG CTG TCC AAT GAC AAC AGG ACC CTC AC - #T CTA CTC AGT GTC        ACA     1248                                                                    Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Th - #r Leu Leu Ser Val Thr                          405  - #               410  - #               415              - - AGG AAT GAT GTA GGA CCC TAT GAG TGT GGA AT - #C CAG AAC GAA TTA AGT         1296                                                                       Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Il - #e Gln Asn Glu Leu Ser                       420      - #           425      - #           430                  - - GTT GAC CAC AGC GAC CCA GTC ATC CTG AAT GT - #C CTC TAT GGC CCA GAC         1344                                                                       Val Asp His Ser Asp Pro Val Ile Leu Asn Va - #l Leu Tyr Gly Pro Asp                   435          - #       440          - #       445                      - - GAC CCC ACC ATT TCC CCC TCA TAC ACC TAT TA - #C CGT CCA GGG GTG AAC         1392                                                                       Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Ty - #r Arg Pro Gly Val Asn               450              - #   455              - #   460                          - - CTC AGC CTC TCC TGC CAT GCA GCC TCT AAC CC - #A CCT GCA CAG TAT TCT         1440                                                                       Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pr - #o Pro Ala Gln Tyr Ser           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TGG CTG ATT GAT GGG AAC ATC CAG CAA CAC AC - #A CAA GAG CTC TTT        ATC     1488                                                                    Trp Leu Ile Asp Gly Asn Ile Gln Gln His Th - #r Gln Glu Leu Phe Ile                          485  - #               490  - #               495              - - TCC AAC ATC ACT GAG AAG AAC AGC GGA CTC TA - #T ACC TGC CAG GCC AAT         1536                                                                       Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Ty - #r Thr Cys Gln Ala Asn                       500      - #           505      - #           510                  - - AAC TCA GCC AGT GGC CAC AGC AGG ACT ACA GT - #C AAG ACA ATC ACA GTC         1584                                                                       Asn Ser Ala Ser Gly His Ser Arg Thr Thr Va - #l Lys Thr Ile Thr Val                   515          - #       520          - #       525                      - - TCT GCG GAG CTG CCC AAG CCC TCC ATC TCC AG - #C AAC AAC TCC AAA CCC         1632                                                                       Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Se - #r Asn Asn Ser Lys Pro               530              - #   535              - #   540                          - - GTG GAG GAC AAG GAT GCT GTG GCC TTC ACC TG - #T GAA CCT GAG GCT CAG         1680                                                                       Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cy - #s Glu Pro Glu Ala Gln           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - AAC ACA ACC TAC CTG TGG TGG GTA AAT GGT CA - #G AGC CTC CCA GTC        AGT     1728                                                                    Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gl - #n Ser Leu Pro Val Ser                          565  - #               570  - #               575              - - CCC AGG CTG CAG CTG TCC AAT GGC AAC AGG AC - #C CTC ACT CTA TTC AAT         1776                                                                       Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Th - #r Leu Thr Leu Phe Asn                       580      - #           585      - #           590                  - - GTC ACA AGA AAT GAC GCA AGA GCC TAT GTA TG - #T GGA ATC CAG AAC TCA         1824                                                                       Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cy - #s Gly Ile Gln Asn Ser                   595          - #       600          - #       605                      - - GTG AGT GCA AAC CGC AGT GAC CCA GTC ACC CT - #G GAT GTC CTC TAT GGG         1872                                                                       Val Ser Ala Asn Arg Ser Asp Pro Val Thr Le - #u Asp Val Leu Tyr Gly               610              - #   615              - #   620                          - - CCG GAC ACC CCC ATC ATT TCC CCC CCA GAC TC - #G TCT TAC CTT TCG GGA         1920                                                                       Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Se - #r Ser Tyr Leu Ser Gly           625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - GCG AAC CTC AAC CTC TCC TGC CAC TCG GCC TC - #T AAC CCA TCC CCG        CAG     1968                                                                    Ala Asn Leu Asn Leu Ser Cys His Ser Ala Se - #r Asn Pro Ser Pro Gln                          645  - #               650  - #               655              - - TAT TCT TGG CGT ATC AAT GGG ATA CCG CAG CA - #A CAC ACA CAA GTT CTC         2016                                                                       Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gl - #n His Thr Gln Val Leu                       660      - #           665      - #           670                  - - TTT ATC GCC AAA ATC ACG CCA AAT AAT AAC GG - #G ACC TAT GCC TGT TTT         2064                                                                       Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gl - #y Thr Tyr Ala Cys Phe                   675          - #       680          - #       685                      - - GTC TCT AAC TTG GCT ACT GGC CGC AAT AAT TC - #C ATA GTC AAG AGC ATC         2112                                                                       Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Se - #r Ile Val Lys Ser Ile               690              - #   695              - #   700                          - - ACA GTC TCT GCA TCT GGA ACT TCT CCT GGT CT - #C TCA GCT GGG GCC ACT         2160                                                                       Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Le - #u Ser Ala Gly Ala Thr           705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - GTC GGC ATC ATG ATT GGA GTG CTG GTT GGG GT - #T GCT CTG ATA                 - #2202                                                                   Val Gly Ile Met Ile Gly Val Leu Val Gly Va - #l Ala Leu Ile                                   725  - #               730                                     - - TAGCAGCCCTGGTGTAGT              - #                  - #                     222 - #0                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 734 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Arg Pro Ala Asp Gln Thr Val Thr Ala Ala Le - #u Thr Lys Arg Ser Trp        1               5 - #                 10 - #                 15              - - Asn Ser Ser Thr Ser Pro Gln Arg Arg Thr Gl - #u Gln Thr Ala Glu Thr                   20     - #             25     - #             30                  - - Met Glu Ser Pro Ser Ala Pro Pro His Arg Tr - #p Cys Ile Pro Trp Gln               35         - #         40         - #         45                      - - Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Ph - #e Trp Asn Pro Pro Thr           50             - #     55             - #     60                          - - Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Ph - #e Asn Val Ala Glu Gly       65                 - # 70                 - # 75                 - # 80       - - Lys Glu Val Leu Leu Leu Val His Asn Leu Pr - #o Gln His Leu Phe Gly                       85 - #                 90 - #                 95              - - Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gl - #y Asn Arg Gln Ile Ile                  100      - #           105      - #           110                  - - Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pr - #o Gly Pro Ala Tyr Ser              115          - #       120          - #       125                      - - Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Le - #u Leu Ile Gln Asn Ile          130              - #   135              - #   140                          - - Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu Hi - #s Val Ile Lys Ser Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Ar - #g Val Tyr Pro Glu        Leu                                                                                             165  - #               170  - #               175             - - Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Ly - #s Pro Val Glu Asp Lys                  180      - #           185      - #           190                  - - Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Th - #r Gln Asp Ala Thr Tyr              195          - #       200          - #       205                      - - Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Va - #l Ser Pro Arg Leu Gln          210              - #   215              - #   220                          - - Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Ph - #e Asn Val Thr Arg Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln As - #n Pro Val Ser Ala        Arg                                                                                             245  - #               250  - #               255             - - Arg Ser Asp Ser Val Ile Leu Asn Val Leu Ty - #r Gly Pro Asp Ala Pro                  260      - #           265      - #           270                  - - Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Se - #r Gly Glu Asn Leu Asn              275          - #       280          - #       285                      - - Leu Ser Cys His Ala Ala Ser Asn Pro Pro Al - #a Gln Tyr Ser Trp Phe          290              - #   295              - #   300                          - - Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Gl - #u Leu Phe Ile Pro Asn      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cy - #s Gln Ala His Asn        Ser                                                                                             325  - #               330  - #               335             - - Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Th - #r Ile Thr Val Tyr Ala                  340      - #           345      - #           350                  - - Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn As - #n Ser Asn Pro Val Glu              355          - #       360          - #       365                      - - Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pr - #o Glu Ile Gln Asn Thr          370              - #   375              - #   380                          - - Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Le - #u Pro Val Ser Pro Arg      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Th - #r Leu Leu Ser Val        Thr                                                                                             405  - #               410  - #               415             - - Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Il - #e Gln Asn Glu Leu Ser                  420      - #           425      - #           430                  - - Val Asp His Ser Asp Pro Val Ile Leu Asn Va - #l Leu Tyr Gly Pro Asp              435          - #       440          - #       445                      - - Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Ty - #r Arg Pro Gly Val Asn          450              - #   455              - #   460                          - - Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pr - #o Pro Ala Gln Tyr Ser      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Trp Leu Ile Asp Gly Asn Ile Gln Gln His Th - #r Gln Glu Leu Phe        Ile                                                                                             485  - #               490  - #               495             - - Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Ty - #r Thr Cys Gln Ala Asn                  500      - #           505      - #           510                  - - Asn Ser Ala Ser Gly His Ser Arg Thr Thr Va - #l Lys Thr Ile Thr Val              515          - #       520          - #       525                      - - Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Se - #r Asn Asn Ser Lys Pro          530              - #   535              - #   540                          - - Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cy - #s Glu Pro Glu Ala Gln      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gl - #n Ser Leu Pro Val        Ser                                                                                             565  - #               570  - #               575             - - Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Th - #r Leu Thr Leu Phe Asn                  580      - #           585      - #           590                  - - Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cy - #s Gly Ile Gln Asn Ser              595          - #       600          - #       605                      - - Val Ser Ala Asn Arg Ser Asp Pro Val Thr Le - #u Asp Val Leu Tyr Gly          610              - #   615              - #   620                          - - Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Se - #r Ser Tyr Leu Ser Gly      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Ala Asn Leu Asn Leu Ser Cys His Ser Ala Se - #r Asn Pro Ser Pro        Gln                                                                                             645  - #               650  - #               655             - - Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gl - #n His Thr Gln Val Leu                  660      - #           665      - #           670                  - - Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gl - #y Thr Tyr Ala Cys Phe              675          - #       680          - #       685                      - - Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Se - #r Ile Val Lys Ser Ile          690              - #   695              - #   700                          - - Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Le - #u Ser Ala Gly Ala Thr      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Val Gly Ile Met Ile Gly Val Leu Val Gly Va - #l Ala Leu Ile                             725  - #               730                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - CCAGTGAATT CCTAATACGA CTACCTATAG GTTAAAACAG C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - GATGAACCCT CGAGACCCAT TATG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CCACCAAGTA CGTAACCACA TATGG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - GTGAGGACTG CTGG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CACCACTGCC CTCGAGAAGC TCACTATTG         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - CACCACTGCC CTCGAGAAGC TCACTATTG         - #                  - #                29                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for stimulating an immune response to animmunogenic protein or fragment thereof, in a subject,comprisingadministering, in a physiologically acceptable carrier, aneffective amount of a composition comprising a recombinant poliovirusnucleic acid having a foreign nucleotide sequence encoding, in anexpressible form, an immunogenic protein or fragment thereof substitutedfor the entire P1 capsid precursor region of the poliovirus genome. 2.The method of claim 1 wherein the recombinant poliovirus nucleic acid isencapsidated.
 3. The method of claim 1 wherein the composition isadministered orally or by intramuscular injections.
 4. The method ofclaim 1 wherein the immunogenic protein or fragment thereof is a humanimmunodeficiency virus type 1 protein or fragment thereof.
 5. The methodof claim 4 wherein the human immunodeficiency virus type 1 protein orfragment thereof is selected from the group consisting of the gagprotein, the pol protein, and the env protein of human immunodeficiencyvirus type
 1. 6. The method of claim 5 wherein the humanimmunodeficiency virus type 1 protein or fragment thereof comprises thehuman immunodeficiency virus type 1 gag protein (SEQ ID NO: 17).
 7. Themethod of claim 1 wherein the immunogenic protein or fragment thereof isa tumor-associated antigen or fragment thereof.
 8. The method of claim 7wherein the tumor-associated antigen is carcinoembryonic antigen.
 9. Amethod for stimulating in a subject an immune response to the gagprotein of the human immunodeficiency virus type 1,comprisingadministering, in a physiologically acceptable carrier, aneffective amount of a composition comprising an encapsidated recombinantpoliovirus nucleic acid having the nucleotide sequence of the humanimmunodeficiency virus type 1 gag gene, in expressible form, substitutedfor the entire P1 capsid precursor region of the poliovirus genome. 10.A method for stimulating in a subject an immune response tocarcinoembryonic antigen, comprisingadministering, in a physiologicallyacceptable carrier, an effective amount of a composition comprising anencapsidated recombinant poliovirus nucleic acid having the nucleotidesequence of the gene encoding the carcinoembryonic antigen, inexpressible form, substituted for the entire P1 capsid precursor regionof the poliovirus genome.